Transporters and ion channels

ABSTRACT

The invention provides human transporters and ion channels (TRICH) and polynucleotides which identify and encode TRICH. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of TRICH.

TECHNICAL FIELD

This invention relates to nucleic acid and amino acid sequences oftransporters and ion channels and to the use of these sequences in thediagnosis, treatment, and prevention of transport, neurological, muscle,and immunological disorders, and in the assessment of the effects ofexogenous compounds on the expression of nucleic acid and amino acidsequences of transporters and ion channels.

BACKGROUND OF THE INVENTION

Eukaryotic cells are surrounded and subdivided into functionallydistinct organelles by hydrophobic lipid bilayer membranes which arehighly impermeable to most polar molecules. Cells and organelles requiretransport proteins to import and export essential nutrients and metalions including K⁺, NH₄ ⁺, P_(i), SO₄ ²⁻, sugars, and vitamins, as wellas various metabolic waste products. Transport proteins also play rolesin antibiotic resistance, toxin secretion, ion balance, synapticneurotransmission, kidney function, intestinal absorption, tumor growth,and other diverse cell functions (Griffith, J. and C. Sansom (1998) TheTransporter Facts Book, Academic Press, San Diego Calif., pp. 3-29).Transport can occur by a passive concentration-dependent mechanism, orcan be linked to an energy source such as ATP hydrolysis or an iongradient. Proteins that function in transport include carrier proteins,which bind to a specific solute and undergo a conformational change thattranslocates the bound solute across the membrane, and channel proteins,which form hydrophilic pores that allow specific solutes to diffusethrough the membrane down an electrochemical solute gradient.

Carrier proteins which transport a single solute from one side of themembrane to the other are called uniporters. In contrast, coupledtransporters link the transfer of one solute with simultaneous orsequential transfer of a second solute, either in the same direction(symport) or in the opposite direction (antiport). For example,intestinal and kidney epithelium contains a variety of symporter systemsdriven by the sodium gradient that exists across the plasma membrane.Sodium moves into the cell down its electrochemical gradient and bringsthe solute into the cell with it. The sodium gradient that provides thedriving force for solute uptake is maintained by the ubiquitous Na⁺/K⁺ATPase system. Sodium-coupled transporters include the mammalian glucosetransporter (SGLT1), iodide transporter (NIS), and multivitamintransporter (SMVT). All three transporters have twelve putativetransmembrane segments, extracellular glycosylation sites, andcytoplasically-oriented N− and C-termini. NIS plays a crucial role inthe evaluation, diagnosis, and treatment of various thyroid pathologiesbecause it is the molecular basis for radioiodide thyroid-imagingtechniques and for specific targeting of radioisotopes to the thyroidgland (Levy, O. et al. (1997) Proc. Natl. Acad. Sci. USA 94:5568-5573).SMVT is expressed in the intestinal mucosa, kidney, and placenta, and isimplicated in the transport of the water-soluble vitamins, e.g., biotinand pantothenate (Prasad, P. D. et al. (1998) J. Biol. Chem.273:7501-7506).

One of the largest families of transporters is the major facilitatorsuperfamily (MFS), also called the uniporter-symporter-antiporterfamily. MFS transporters are single polypeptide carriers that transportsmall solutes in response to ion gradients. Members of the MFS are foundin all classes of living organisms, and include transporters for sugars,oligosaccharides, phosphates, nitrates, nucleosides, monocarboxylates,and drugs. MFS transporters found in eukaryotes all have a structurecomprising 12 transmembrane segments (Pao, S. S. et al. (1998)Microbiol. Molec. Biol. Rev. 62:1-34). The largest family of MFStransporters is the sugar transporter family, which includes the sevenglucose transporters (GLUT1-GLUT7) found in humans that are required forthe transport of glucose and other hexose sugars. These glucosetransport proteins have unique tissue distributions and physiologicalfunctions. GLUT1 provides many cell types with their basal glucoserequirements and transports glucose across epithelial and endothelialbarrier tissues; GLUT2 facilitates glucose uptake or efflux from theliver; GLUT3 regulates glucose supply to neurons; GLUT4 is responsiblefor insulin-regulated glucose disposal; and GLUT5 regulates fructoseuptake into skeletal muscle. Defects in glucose transporters areinvolved in a recently identified neurological syndrome causinginfantile seizures and developmental delay, as well as glycogen storagedisease, Fanconi-Bickel syndrome, and non-insulin-dependent diabetesmellitus (Mueckler, M. (1994) Eur. J. Biochem. 219:713-725; Longo, N.and L. J. Elsas (1998) Adv. Pediatr. 45:293-313).

Monocarboxylate anion transporters are proton-coupled symporters with abroad substrate specificity that includes L-lactate, pyruvate, and theketone bodies acetate, acetoacetate, and beta-hydroxybutyrate. At leastseven isoforms have been identified to date. The isoforms are predictedto have twelve transmembrane (TM) helical domains with a largeintracellular loop between TM6 and TM7, and play a critical role inmaintaining intracellular pH by removing the protons that are producedstoichiometrically with lactate during glycolysis. The bestcharacterized H⁺-monocarboxylate transporter is that of the erythrocytemembrane, which transports L-lactate and a wide range of other aliphaticmonocarboxylates. Other cells possess H⁺-linked monocarboxylatetransporters with differing substrate and inhibitor selectivities. Inparticular, cardiac muscle and tumor cells have transporters that differin their K_(m) values for certain substrates, includingstereoselectivity for L- over D-lactate, and in their sensitivity toinhibitors. There are Na⁺-monocarboxylate cotransporters on the luminalsurface of intestinal and kidney epithelia, which allow the uptake oflactate, pyruvate, and ketone bodies in these tissues. In addition,there are specific and selective transporters for organic cations andorganic anions in organs including the kidney, intestine and liver.Organic anion transporters are selective for hydrophobic, chargedmolecules with electron-attracting side groups. Organic cationtransporters, such as the ammonium transporter, mediate the secretion ofa variety of drugs and endogenous metabolites, and contribute to themaintenance of intercellular pH (Poole, R. C. and A. P. Halestrap (1993)Am. J. Physiol. 264:C761-C782; Price, N. T. et al. (1998) Biochem. J.329:321-328; and Martinelle, K. and I. Haggstrom (1993) J. Biotechnol.30:339-350).

ATP-binding cassette (ABC) transporters are members of a superfamily ofmembrane proteins that transport substances ranging from small moleculessuch as ions, sugars, amino acids, peptides, and phospholipids, tolipopeptides, large proteins, and complex hydrophobic drugs. ABCtransporters consist of four modules: two nucleotide-binding domains(NBD), which hydrolyze ATP to supply the energy required for transport,and two membrane-spanning domains (MSD), each containing six putativetransmembrane segments. These four modules may be encoded by a singlegene, as is the case for the cystic fibrosis transmembrane regulator(CFTR), or by separate genes. When encoded by separate genes, each geneproduct contains a single NBD and MSD. These “half-molecules” form homo-and heterodimers, such as Tap1 and Tap2, the endoplasmic reticulum-basedmajor histocompatibility (MHC) peptide transport system. Several geneticdiseases are attributed to defects in ABC transporters, such as thefollowing diseases and their corresponding proteins: cystic fibrosis(CFIR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophyprotein, ALDP), Zellweger syndrome (peroxisomal membrane protein-70,PMP70), and hyperinsulinemic hypoglycemia (sulfonylurea receptor, SUR).Overexpression of the multidrug resistance (MDR) protein, another ABCtransporter, in human cancer cells makes the cells resistant to avariety of cytotoxic drugs used in chemotherapy (Taglicht, D. and S.Michaelis (1998) Meth. Enzymol. 292:130-162).

A number of metal ions such as iron, zinc, copper, cobalt, manganese,molybdenum, selenium, nickel, and chromium are important as cofactorsfor a number of enzymes. For example, copper is involved in hemoglobinsynthesis, connective tissue metabolism, and bone development, by actingas a cofactor in oxidoreductases such as superoxide dismutase,ferroxidase (ceruloplasmin), and lysyl oxidase. Copper and other metalions must be provided in the diet, and are absorbed by transporters inthe gastrointestinal tract. Plasma proteins transport the metal ions tothe liver and other target organs, where specific transporters move theions into cells and cellular organelles as needed. Imbalances in metalion metabolism have been associated with a number of disease states(Danks, D. M. (1986) J. Med. Genet. 23:99-106).

Transport of fatty acids across the plasma membrane can occur bydiffusion, a high capacity, low affinity process. However, under normalphysiological conditions a significant fraction of fatty acid transportappears to occur via a high affinity, low capacity protein-mediatedtransport process. Fatty acid transport protein (FATP), an integralmembrane protein with four transmembrane segments, is expressed intissues exhibiting high levels of plasma membrane fatty acid flux, suchas muscle, heart, and adipose. Expression of FATP is upregulated in3T3-L1 cells during adipose conversion, and expression in COS7fibroblasts elevates uptake of long-chain fatty acids (Hui, T. Y. et al.(1998) J. Biol. Chem. 273:27420-27429).

Mitochondrial carrier proteins are transmembrane-spanning proteins whichtransport ions and charged metabolites between the cytosol and themitochondrial matrix. Examples include the ADP, ATP carrier protein; the2-oxoglutarate/malate carrier; the phosphate carrier protein; thepyruvate carrier; the dicarboxylate carrier which transports malate,succinate, fumarate, and phosphate; the tricarboxylate carrier whichtransports citrate and malate; and the Grave's disease carrier protein,a protein recognized by IgG in patients with active Grave's disease, anautoimmune disorder resulting in hyperthyroidism. Proteins in thisfamily consist of three tandem repeats of an approximately 100 aminoacid domain, each of which contains two transmembrane regions (Stryer,L. (1995) Biochemistry, W. H. Freeman and Company, New York N.Y., p.551; PROSITE PDOC00189 Mitochondrial energy transfer proteins signature;Online Mendelian Inheritance in Man (OMIM) *275000 Graves Disease).

This class of transporters also includes the mitochondrial uncouplingproteins, which create proton leaks across the inner mitochondrialmembrane, thus uncoupling oxidative phosphorylation from ATP synthesis.The result is energy dissipation in the form of heat. Mitochondrialuncoupling proteins have been implicated as modulators ofthermoregulation and metabolic rate, and have been proposed as potentialtargets for drugs against metabolic diseases such as obesity (Ricquier,D. et al. (1999) J. Int. Med. 245:637-642).

Ion Channels

The electrical potential of a cell is generated and maintained bycontrolling the movement of ions across the plasma membrane. Themovement of ions requires ion channels, which form ion-selective poreswithin the membrane. There are two basic types of ion channels, iontransporters and gated ion channels. Ion transporters utilize the energyobtained from ATP hydrolysis to actively transport an ion against theion's concentration gradient. Gated ion channels allow passive flow ofan ion down the ion's electrochemical gradient under restrictedconditions. Together, these types of ion channels generate, maintain,and utilize an electrochemical gradient that is used in 1) electricalimpulse conduction down the axon of a nerve cell, 2) transport ofmolecules into cells against concentration gradients, 3) initiation ofmuscle contraction, and 4) endocrine cell secretion.

Ion Transporters

Ion transporters generate and maintain the resting electrical potentialof a cell. Utilizing the energy derived from ATP hydrolysis, theytransport ions against the ion's concentration gradient. Thesetransmembrane ATPases are divided into three families. Thephosphorylated (P) class ion transporters, including Na⁺—K⁺ ATPase,Ca²⁺-ATPase, and H⁺-ATPase, are activated by a phosphorylation event.P-class ion transporters are responsible for maintaining restingpotential distributions such that cytosolic concentrations of Na⁺ andCa²⁺ are low and cytosolic concentration of K⁺ is high. The vacuolar (V)class of ion transporters includes H⁺ pumps on intracellular organelles,such as lysosomes and Golgi. V-class ion transporters are responsiblefor generating the low pH within the lumen of these organelles that isrequired for function. The coupling factor (F) class consists of H⁺pumps in the mitochondria. F-class ion transporters utilize a protongradient to generate ATP from ADP and inorganic phosphate (P_(i)).

The P-ATPases are hexamers of a 100 kD subunit with ten transmembranedomains and several large cytoplasmic regions that may play a role inion binding (Scarborough, G. A. (1999) Curr. Opin. Cell Biol.11:517-522). The V-ATPases are composed of two functional domains: theV₁ domain, a peripheral complex responsible for ATP hydrolysis; and theV₀ domain, an integral complex responsible for proton translocationacross the membrane. The F-ATPases are structurally and evolutionarilyrelated to the V-ATPases. The F-ATPase F₀ domain contains 12 copies ofthe c subunit, a highly hydrophobic protein composed of twotransmembrane domains and containing a single buried carboxyl group inTM2 that is essential for proton transport. The V-ATPase V₀ domaincontains three types of homologous c subunits with four or fivetransmembrane domains and the essential carboxyl group in TM4 or TM3.Both types of complex also contain a single a subunit that may beinvolved in regulating the pH dependence of activity (Forgac, M. (1999)J. Biol. Chem. 274:12951-12954).

The resting potential of the cell is utilized in many processesinvolving carrier proteins and gated ion channels. Carrier proteinsutilize the resting potential to transport molecules into and out of thecell. Amino acid and glucose transport into many cells is linked tosodium ion co-transport (symport) so that the movement of Na⁺ down anelectrochemical gradient drives transport of the other molecule up aconcentration gradient. Similarly, cardiac muscle links transfer of Ca²⁺out of the cell with transport of Na⁺ into the cell (antiport).

Gated Ion Channels

Gated ion channels control ion flow by regulating the opening andclosing of pores. The ability to control ion flux through various gatingmechanisms allows ion channels to mediate such diverse signaling andhomeostatic functions as neuronal and endocrine signaling, musclecontraction, fertilization, and regulation of ion and pH balance. Gatedion channels are categorized according to the manner of regulating thegating function. Mechanically-gated channels open their pores inresponse to mechanical stress; voltage-gated channels (e.g., Na⁺, K⁺,Ca²⁺, and Cl⁻ channels) open their pores in response to changes inmembrane potential; and ligand-gated channels (e.g., acetylcholine-,serotonin-, and glutamate-gated cation channels, and GABA- andglycine-gated chloride channels) open their pores in the presence of aspecific ion, nucleotide, or neurotransmitter. The gating properties ofa particular ion channel (i.e., its threshold for and duration ofopening and closing) are sometimes modulated by association withauxiliary channel proteins and/or post translational modifications, suchas phosphorylation.

Mechanically-gated or mechanosensitive ion channels act as transducersfor the senses of touch, hearing, and balance, and also play importantroles in cell volume regulation, smooth muscle contraction, and cardiacrhythm generation. A stretch-inactivated channel (SIC) was recentlycloned from rat kidney. The SIC channel belongs to a group of channelswhich are activated by pressure or stress on the cell membrane andconduct both Ca²⁺ and Na⁺ (Suzuki, M. et al. (1999) J. Biol. Chem.274:6330-6335).

The pore-forming subunits of the voltage-gated cation channels form asuperfamily of ion channel proteins. The characteristic domain of thesechannel proteins comprises six transmembrane domains (S1-S6), apore-forming region (P) located between S5 and S6, and intracellularamino and carboxy termini. In the Na⁺ and Ca²⁺ subfamilies, this domainis repeated four times, while in the K⁺ channel subfamily, each channelis formed from a tetramer of either identical or dissimilar subunits.The P region contains information specifying the ion selectivity for thechannel. In the case of K⁺ channels, a GYG tripeptide is involved inthis selectivity (Ishii, T. M. et al. (1997) Proc. Natl. Acad. Sci. USA94:11651-11656).

Voltage-gated Na⁺ and K⁺ channels are necessary for the function ofelectrically excitable cells, such as nerve and muscle cells. Actionpotentials, which lead to neurotransmitter release and musclecontraction, arise from large, transient changes in the permeability ofthe membrane to Na⁺ and K⁺ ions. Depolarization of the membrane beyondthe threshold level opens voltage-gated Na⁺ channels. Sodium ions flowinto the cell, further depolarizing the membrane and opening morevoltage-gated Na⁺ channels, which propagates the depolarization down thelength of the cell. Depolarization also opens voltage-gated potassiumchannels. Consequently, potassium ions flow outward, which leads torepolarization of the membrane. Voltage-gated channels utilize chargedresidues in the fourth transmembrane segment (S4) to sense voltagechange. The open state lasts only about 1 millisecond, at which time thechannel spontaneously converts into an inactive state that cannot beopened irrespective of the membrane potential. Inactivation is mediatedby the channel's N-terminus, which acts as a plug that closes the pore.The transition from an inactive to a closed state requires a return toresting potential.

Voltage-gated Na⁺ channels are heterotrimeric complexes composed of a260 kDa pore-forming α a subunit that associates with two smallerauxiliary subunits, β1 and β2. The β2 subunit is a integral membraneglycoprotein that contains an extracellular Ig domain, and itsassociation with α and β1 subunits correlates with increased functionalexpression of the channel, a change in its gating properties, as well asan increase in whole cell capacitance due to an increase in membranesurface area (Isom, L. L. et al. (1995) Cell 83:433-442).

Non voltage-gated Na⁺ channels include the members of theamiloride-sensitive Na⁺ channel/degenerin (NaC/DEG) family. Channelsubunits of this family are thought to consist of two transmembranedomains flanking a long extracellular loop, with the amino and carboxyltermini located within the cell. The NaC/DEG family includes theepithelial Na⁺ channel (ENaC) involved in Na⁺ reabsorption in epitheliaincluding the airway, distal colon, cortical collecting duct of thekidney, and exocrine duct glands. Mutations in ENaC result inpseudohypoaldosteronism type 1 and Liddle's syndrome(pseudohyperaldosteronism). The NaC/DEG family also includes therecently characterized H⁺-gated cation channels or acid-sensing ionchannels (ASIC). ASIC subunits are expressed in the brain and formheteromultimeric Na⁺-permeable channels. These channels require acid pHfluctuations for activation. ASIC subunits show homology to thedegenerins, a family of mechanically-gated channels originally isolatedfrom C. elegans. Mutations in the degenerins cause neurodegeneration.ASIC subunits may also have a role in neuronal function, or in painperception, since tissue acidosis causes pain (Waldmann, R. and M.Lazdunski (1998) Curr. Opin. Neurobiol. 8:418-424; Eglen, R. M. et al.(1999) Trends Pharmacol. Sci. 20:337-342).

K⁺ channels are located in all cell types, and may be regulated byvoltage, ATP concentration, or second messengers such as Ca²⁺ and cAMP.In non-excitable tissue, K⁺ channels are involved in protein synthesis,control of endocrine secretions, and the maintenance of osmoticequilibrium across membranes. In neurons and other excitable cells, inaddition to regulating action potentials and repolarizing membranes, K⁺channels are responsible for setting resting membrane potential. Thecytosol contains non-diffusible anions and, to balance this net negativecharge, the cell contains a Na⁺—K⁺ pump and ion channels that providethe redistribution of Na⁺, K⁺, and Cl⁻. The pump actively transports Na⁺out of the cell and K⁺ into the cell in a 3:2 ratio. Ion channels in theplasma membrane allow K⁺ and Cl⁻ to flow by passive diffusion. Becauseof the high negative charge within the cytosol, Cl⁻ flows out of thecell. The flow of K⁺ is balanced by an electromotive force pulling K⁺into the cell, and a K⁺ concentration gradient pushing K⁺ out of thecell. Thus, the resting membrane potential is primarily regulated by K⁺flow (Salkoff, L. and T. Jegla (1995) Neuron 15:489-492).

Potassium channel subunits of the Shaker-like superfamily all have thecharacteristic six transmembrane/1 pore domain structure. Four subunitscombine as homo- or heterotetramers to form functional K channels. Thesepore-forming subunits also associate with various cytoplasmic β subunitsthat alter channel inactivation kinetics. The Shaker-like channel familyincludes the voltage-gated K⁺ channels as well as the delayed rectifiertype channels such as the human ether-a-go-go related gene (HERG)associated with long QT, a cardiac dysrythmia syndrome (Curran, M. E.(1998) Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G. J. and M. L.Garcia (1999) Curr. Opin. Chem. Biol. 3:448-458).

A second superfamily of K⁺ channels is composed of the inward rectifyingchannels (Kir). Kir channels have the property of preferentiallyconducting K⁺ currents in the inward direction. These proteins consistof a single potassium selective pore domain and two transmembranedomains, which correspond to the fifth and sixth transmembrane domainsof voltage-gated K⁺ channels. Kir subunits also associate as tetramers.The Kir family includes ROMK1, mutations in which lead to Barttersyndrome, a renal tubular disorder. Kir channels are also involved inregulation of cardiac pacemaker activity, seizures and epilepsy, andinsulin regulation (Doupnik, C. A. et al. (1995) Curr. Opin. Neurobiol.5:268-277; Curran, supra).

The recently recognized TWIK K⁺ channel family includes the mammalianTWIK-1, TREK-1 and TASK proteins. Members of this family possess anoverall structure with four transmembrane domains and two P domains.These proteins are probably involved in controlling the restingpotential in a large set of cell types (Duprat, F. et al. (1997) EMBO J16:5464-5471).

The voltage-gated Ca²⁺ channels have been classified into severalsubtypes based upon their electrophysiological and pharmacologicalcharacteristics. L-type Ca²⁺ channels are predominantly expressed inheart and skeletal muscle where they play an essential role inexcitation-contraction coupling. T-type channels are important forcardiac pacemaker activity, while N-type and P/Q-type channels areinvolved in the control of neurotransmitter release in the central andperipheral nervous system. The L-type and N-type voltage-gated Ca²⁺channels have been purified and, though their functions differdramatically, they have similar subunit compositions. The channels arecomposed of three subunits. The α₁ subunit forms the membrane pore andvoltage sensor, while the α₂δ and β subunits modulate thevoltage-dependence, gating properties, and the current amplitude of thechannel. These subunits are encoded by at least six α₁ , one α₂δ, andfour β genes. A fourth subunit, γ, has been identified in skeletalmuscle (Walker, D. et al. (1998) J. Biol. Chem. 273:2361-2367;McCleskey, E. W. (1994) Curr. Opin. Neurobiol. 4:304-312).

Chloride channels are necessary in endocrine secretion and in regulationof cytosolic and organelle pH. In secretory epithelial cells, Cl⁻ entersthe cell across a basolateral membrane through an Na⁺, K⁺/Cl⁻cotransporter, accumulating in the cell above its electrochemicalequilibrium concentration. Secretion of Cl⁻ from the apical surface, inresponse to hormonal stimulation, leads to flow of Na⁺ and water intothe secretory lumen. The cystic fibrosis transmembrane conductanceregulator (CFTR) is a chloride channel encoded by the gene for cysticfibrosis, a common fatal genetic disorder in humans. CFIR is a member ofthe ABC transporter family, and is composed of two domains eachconsisting of six transmembrane domains followed by a nucleotide-bindingsite. Loss of CFTR function decreases transepithelial water secretionand, as a result, the layers of mucus that coat the respiratory tree,pancreatic ducts, and intestine are dehydrated and difficult to clear.The resulting blockage of these sites leads to pancreatic insufficiency,“meconium ileus”, and devastating “chronic obstructive pulmonarydisease” (Al-Awqati, Q. et al. (1992) J. Exp. Biol. 172:245-266).

The voltage-gated chloride channels (CLC) are characterized by 10-12transmembrane domains, as well as two small globular domains known asCBS domains. The CLC subunits probably function as homotetramers. CLCproteins are involved in regulation of cell volume, membrane potentialstabilization, signal transduction, and transepithelial transport.Mutations in CLC-1, expressed predominantly in skeletal muscle, areresponsible for autosomal recessive generalized myotonia and autosomaldominant myotonia congenita, while mutations in the kidney channel CLC-5lead to kidney stones (Jentsch, T. J. (1996) Curr. Opin. Neurobiol.6:303-310).

Ligand-gated channels open their pores when an extracellular orintracellular mediator binds to the channel. Neurotransmitter-gatedchannels are channels that open when a neurotransmitter binds to theirextracellular domain. These channels exist in the postsynaptic membraneof nerve or muscle cells. There are two types of neurotransmitter-gatedchannels. Sodium channels open in response to excitatoryneurotransmitters, such as acetylcholine, glutamate, and serotonin. Thisopening causes an influx of Na⁺ and produces the initial localizeddepolarization that activates the voltage-gated channels and starts theaction potential. Chloride channels open in response to inhibitoryneurotransmitters, such as γ-aminobutyric acid (GABA) and glycine,leading to hyperpolarization of the membrane and the subsequentgeneration of an action potential. Neurotransmitter-gated ion channelshave four transmembrane domains and probably function as pentamers(Jentsch, supra). Amino acids in the second transmembrane domain appearto be important in determining channel permeation and selectivity(Sather, W. A. et al. (1994) Curr. Opin. Neurobiol. 4:313-323).

Ligand-gated channels can be regulated by intracellular secondmessengers. For example, calcium-activated K⁺ channels are gated byinternal calcium ions. In nerve cells, an influx of calcium duringdepolarization opens K⁺ channels to modulate the magnitude of the actionpotential (Ishi et al., supra). The large conductance (BK) channel hasbeen purified from brain and its subunit composition determined. The αsubunit of the BK channel has seven rather than six transmembranedomains in contrast to voltage-gated K⁺ channels. The extratransmembrane domain is located at the subunit N-terminus. A28-amino-acid stretch in the C-terminal region of the subunit (the“calcium bowl” region) contains many negatively charged residues and isthought to be the region responsible for calcium binding The β subunitconsists of two transmembrane domains connected by a glycosylatedextracellular loop, with intracellular N- and C-termini (Kaczorowski,supra; Vergara, C. et al. (1998) Curr. Opin. Neurobiol. 8:321-329).

Cyclic nucleotide-gated (CNG) channels are gated by cytosolic cyclicnucleotides. The best examples of these are the cAMP-gated Na+ channelsinvolved in olfaction and the cGMP-gated cation channels involved invision. Both systems involve ligand-mediated activation of a G-proteincoupled receptor which then alters the level of cyclic nucleotide withinthe cell. CNG channels also represent a major pathway for Ca²⁺ entryinto neurons, and play roles in neuronal development and plasticity. CNGchannels are tetramers containing at least two types of subunits, an αsubunit which can form functional homomeric channels, and a β subunit,which modulates the channel properties. All CNG subunits have sixtransmembrane domains and a pore forming region between the fifth andsixth transmembrane domains, similar to voltage-gated K⁺ channels. Alarge C-terminal domain contains a cyclic nucleotide binding domain,while the N-terminal domain confers variation among channel subtypes(Zufall, F. et al. (1997) Curr. Opin. Neurobiol. 7:404-412).

The activity of other types of ion channel proteins may also bemodulated by a variety of intracellular signalling proteins. Manychannels have sites for phosphorylation by one or more protein kinasesincluding protein kinase A, protein kinase C, tyrosine kinase, andcasein kinase II, all of which regulate ion channel activity in cells.Kir channels are activated by the binding of the Gβγ subunits ofheterotrimeric G-proteins (Reimann, F. and F. M. Ashcroft (1999) Curr.Opin. Cell. Biol. 11:503-508). Other proteins are involved in thelocalization of ion channels to specific sites in the cell membrane.Such proteins include the PDZ domain proteins known as MAGUKs(membrane-associated guanylate kinases) which regulate the clustering ofion channels at neuronal synapses (Craven, S. E. and D. S. Bredt (1998)Cell 93:495-498).

Disease Correlation

The etiology of numerous human diseases and disorders can be attributedto defects in the transport of molecules across membranes. Defects inthe trafficking of membrane-bound transporters and ion channels areassociated with several disorders, e.g., cystic fibrosis,glucose-galactose malabsorption syndrome, hypercholesterolemia, vonGierke disease, and certain forms of diabetes mellitus. Single-genedefect diseases resulting in an inability to transport small moleculesacross membranes include, e.g., cystinuria, iminoglycinuria, Hartupdisease, and Fanconi disease (van't Hoff, W. G. (1996) Exp. Nephrol.4:253-262; Talente, G. M. et al. (1994) Ann. Intern. Med. 120:218-226;and Chillon, M. et al. (1995) New Engl. J. Med. 332:1475-1480).

Human diseases caused by mutations in ion channel genes includedisorders of skeletal muscle, cardiac muscle, and the central nervoussystem. Mutations in the pore-forming subunits of sodium and chloridechannels cause myotonia, a muscle disorder in which relaxation aftervoluntary contraction is delayed. Sodium channel myotonias have beentreated with channel blockers. Mutations in muscle sodium and calciumchannels cause forms of periodic paralysis, while mutations in thesarcoplasmic calcium release channel, T-tubule calcium channel, andmuscle sodium channel cause malignant hyperthermia. Cardiac arrythmiadisorders such as the long QT syndromes and idiopathic ventricularfibrillation are caused by mutations in potassium and sodium channels(Cooper, E. C. and L. Y. Jan (1998) Proc. Natl. Acad. Sci. USA96:4759-4766). All four known human idiopathic epilepsy genes code forion channel proteins (Berkovic, S. F. and I. E. Scheffer (1999) Curr.Opin. Neurology 12:177-182). Other neurological disorders such asataxias, hemiplegic migraine and hereditary deafness can also resultfrom mutations in ion channel genes (Jen, J. (1999) Curr. Opin.Neurobiol. 9:274-280; Cooper, supra).

Ion channels have been the target for many drug therapies.Neurotransmitter-gated channels have been targeted in therapies fortreatment of insomnia, anxiety, depression, and schizophrenia.Voltage-gated channels have been targeted in therapies for arrhythmia,ischemic stroke, head trauma, and neurodegenerative disease (Taylor, C.P. and L. S. Narasimhan (1997) Adv. Pharmacol. 39:47-98). Variousclasses of ion channels also play an important role in the perception ofpain, and thus are potential targets for new analgesics. These includethe vanilloid-gated ion channels, which are activated by the vanilloidcapsaicin, as well as by noxious heat. Local anesthetics such aslidocaine and mexiletine which blockade voltage-gated Na+ channels havebeen useful in the treatment of neuropathic pain (Eglen, supra).

Ion channels in the immune system have recently been suggested astargets for immunomodulation. T-cell activation depends upon calciumsignaling, and a diverse set of T-cell specific ion channels has beencharacterized that affect this signaling process. Channel blockingagents can inhibit secretion of lymphokines, cell proliferation, andkilling of target cells. A peptide antagonist of the T-cell potassiumchannel Kv1.3 was found to suppress delayed-type hypersensitivity andallogenic responses in pigs, validating the idea of channel blockers assafe and efficacious immunosuppressants (Cahalan, M. D. and K. G. Chandy(1997) Curr. Opin. Biotechnol. 8:749-756).

The discovery of new transporters and ion channels and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention, andtreatment of transport, neurological, muscle, and immunologicaldisorders, and in the assessment of the effects of exogenous compoundson the expression of nucleic acid and amino acid sequences oftransporters and ion channels.

SUMMARY OF THE INVENTION

The invention features purified polypeptides, transporters and ionchannels, referred to collectively as “TRICH” and individually as“TRICH-1,” “TRICH-2,” “TRICH-3,” “TRICH4” “TRICH-5,” “TRICH-6,”“TRICH-7,” “TRICH-8,” “TRICH-9,” “TRICH-10,” “TRICH-11,” “TRICH-12,” and“TRICH-13.” In one aspect, the invention provides an isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of a) an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13, b) a naturally occurring amino acidsequence having at least 90% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-13, c) a biologicallyactive fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an aminoacid sequence selected from the group consisting of SEQ ID NO:1-13. Inone alternative, the invention provides an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:1-13.

The invention further provides an isolated polynucleotide encoding apolypeptide comprising an amino acid sequence selected from the groupconsisting of a) an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13, b) a naturally occurring amino acidsequence having at least 90% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-13, c) a biologicallyactive fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an aminoacid sequence selected from the group consisting of SEQ ID NO:1-13. Inone alternative, the polynucleotide encodes a polypeptide selected fromthe group consisting of SEQ ID NO:1-13. In another alternative, thepolynucleotide is selected from the group consisting of SEQ ID NO:14-26.

Additionally, the invention provides a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding a polypeptide comprising an amino acid sequence selected fromthe group consisting of a) an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-13, b) a naturally occurring amino acidsequence having at least 90% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-13, c) a biologicallyactive fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an aminoacid sequence selected from the group consisting of SEQ ID NO:1-13. Inone alternative, the invention provides a cell transformed with therecombinant polynucleotide. In another alternative, the inventionprovides a transgenic organism comprising the recombinantpolynucleotide.

The invention also provides a method for producing a polypeptidecomprising an amino acid sequence selected from the group consisting ofa) an amino acid sequence selected from the group consisting of SEQ IDNO:1-13, b) a naturally occurring amino acid sequence having at least90% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13, c) a biologically active fragment of anamino acid sequence selected from the group consisting of SEQ IDNO:1-13, and d) an immunogenic fragment of an amino acid sequenceselected from the group consisting of SEQ ID NO:1-13. The methodcomprises a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide comprising a promoter sequence operably linked to apolynucleotide encoding the polypeptide, and b) recovering thepolypeptide so expressed.

Additionally, the invention provides an isolated antibody whichspecifically binds to a polypeptide comprising an amino acid sequenceselected from the group consisting of a) an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-13, b) a naturally occurringamino acid sequence having at least 90% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NO:1-13, c) abiologically active fragment of an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of anamino acid sequence selected from the group consisting of SEQ IDNO:1-13.

The invention further provides an isolated polynucleotide comprising apolynucleotide sequence selected from the group consisting of a) apolynucleotide sequence selected from the group consisting of SEQ IDNO:14-26, b) a naturally occurring polynucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:14-26, c) a polynucleotide sequencecomplementary to a), d) a polynucleotide sequence complementary to b),and e) an RNA equivalent of a)-d). In one alternative, thepolynucleotide comprises at least 60 contiguous nucleotides.

Additionally, the invention provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of a) a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:14-26, b) a naturally occurringpolynucleotide sequence having at least 90% sequence identity to apolynucleotide sequence selected from the group consisting of SEQ IDNO:14-26, c) a polynucleotide sequence complementary to a), d) apolynucleotide sequence complementary to b), and e) an RNA equivalent ofa)-d). The method comprises a) hybridizing the sample with a probecomprising at least 20 contiguous nucleotides comprising a sequencecomplementary to said target polynucleotide in the sample, and whichprobe specifically hybridizes to said target polynucleotide, underconditions whereby a hybridization complex is formed between said probeand said target polynucleotide or fragments thereof, and b) detectingthe presence or absence of said hybridization complex, and optionally,if present, the amount thereof. In one alternative, the probe comprisesat least 60 contiguous nucleotides.

The invention further provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of a) a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:14-26, b) a naturally occurringpolynucleotide sequence having at least 90% sequence identity to apolynucleotide sequence selected from the group consisting of SEQ IDNO:14-26, c) a polynucleotide sequence complementary to a), d) apolynucleotide sequence complementary to b), and e) an RNA equivalent ofa)-d). The method comprises a) amplifying said target polynucleotide orfragment thereof using polymerase chain reaction amplification, and b)detecting the presence or absence of said amplified targetpolynucleotide or fragment thereof, and, optionally, if present, theamount thereof.

The invention further provides a composition comprising an effectiveamount of a polypeptide comprising an amino acid sequence selected fromthe group consisting of a) an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-13, b) a naturally occurring amino acidsequence having at least 90% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-13, c) a biologicallyactive fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an aminoacid sequence selected from the group consisting of SEQ ID NO:1-13, anda pharmaceutically acceptable excipient. In one embodiment, thecomposition comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13. The invention additionally provides amethod of treating a disease or condition associated with decreasedexpression of functional TRICH, comprising administering to a patient inneed of such treatment the composition.

The invention also provides a method for screening a compound foreffectiveness as an agonist of a polypeptide comprising an amino acidsequence selected from the group consisting of a) an amino acid sequenceselected from the group consisting of SEQ ID NO:1-13, b) a naturallyoccurring amino acid sequence having at least 90% sequence identity toan amino acid sequence selected from the group consisting of SEQ IDNO:1-13, c) a biologically active fragment of an amino acid sequenceselected from the group consisting of SEQ ID NO:1-13, and d) animmunogenic fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13. The method comprises a) exposing a samplecomprising the polypeptide to a compound, and b) detecting agonistactivity in the sample. In one alternative, the invention provides acomposition comprising an agonist compound identified by the method anda pharmaceutically acceptable excipient. In another alternative, theinvention provides a method of treating a disease or conditionassociated with decreased expression of functional TRICH, comprisingadministering to a patient in need of such treatment the composition.

Additionally, the invention provides a method for screening a compoundfor effectiveness as an antagonist of a polypeptide comprising an aminoacid sequence selected from the group consisting of a) an amino acidsequence selected from the group consisting of SEQ ID NO:1-13, b) anaturally occurring amino acid sequence having at least 90% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:1-13, c) a biologically active fragment of an amino acidsequence selected from the group consisting of SEQ ID NO:1-13, and d) animmunogenic fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13. The method comprises a) exposing a samplecomprising the polypeptide to a compound, and b) detecting antagonistactivity in the sample. In one alternative, the invention provides acomposition comprising an antagonist compound identified by the methodand a pharmaceutically acceptable excipient. In another alternative, theinvention provides a method of treating a disease or conditionassociated with overexpression of functional TRICH, comprisingadministering to a patient in need of such treatment the composition.

The invention further provides a method of screening for a compound thatspecifically binds to a polypeptide comprising an amino acid sequenceselected from the group consisting of a) an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-13, b) a naturally occurringamino acid sequence having at least 90% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NO:1-13, c) abiologically active fragment of an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of anamino acid sequence selected from the group consisting of SEQ IDNO:1-13. The method comprises a) combining the polypeptide with at leastone test compound under suitable conditions, and b) detecting binding ofthe polypeptide to the test compound, thereby identifying a compoundthat specifically binds to the polypeptide.

The invention further provides a method of screening for a compound thatmodulates the activity of a polypeptide comprising an amino acidsequence selected from the group consisting of a) an amino acid sequenceselected from the group consisting of SEQ ID NO:1-13, b) a naturallyoccurring amino acid sequence having at least 90% sequence identity toan amino acid sequence selected from the group consisting of SEQ IDNO:1-13, c) a biologically active fragment of an amino acid sequenceselected from the group consisting of SEQ ID NO:1-13, and d) animmunogenic fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13. The method comprises a) combining thepolypeptide with at least one test compound under conditions permissivefor the activity of the polypeptide, b) assessing the activity of thepolypeptide in the presence of the test compound, and c) comparing theactivity of the polypeptide in the presence of the test compound withthe activity of the polypeptide in the absence of the test compound,wherein a change in the activity of the polypeptide in the presence ofthe test compound is indicative of a compound that modulates theactivity of the polypeptide.

The invention further provides a method for screening a compound foreffectiveness in altering expression of a target polynucleotide, whereinsaid target polynucleotide comprises a sequence selected from the groupconsisting of SEQ ID NO:14-26, the method comprising a) exposing asample comprising the target polynucleotide to a compound, and b)detecting altered expression of the target polynucleotide.

The invention further provides a method for assessing toxicity of a testcompound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide comprising apolynucleotide sequence selected from the group consisting of i) apolynucleotide sequence selected from the group consisting of SEQ IDNO:14-26, ii) a naturally occurring polynucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:14-26, iii) a polynucleotide sequencecomplementary to i), iv) a polynucleotide sequence complementary to ii),and v) an RNA equivalent of i)-iv). Hybridization occurs underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of i) a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:14-26, ii) a naturally occurringpolynucleotide sequence having at least 90% sequence identity to apolynucleotide sequence selected from the group consisting of SEQ IDNO:14-26, iii) a polynucleotide sequence complementary to i), iv) apolynucleotide sequence complementary to ii), and v) an RNA equivalentof i)-iv). Alternatively, the target polynucleotide comprises a fragmentof a polynucleotide sequence selected from the group consisting of i)-v)above; c) quantifying the amount of hybridization complex; and d)comparing the amount of hybridization complex in the treated biologicalsample with the amount of hybridization complex in an untreatedbiological sample, wherein a difference in the amount of hybridizationcomplex in the treated biological sample is indicative of toxicity ofthe test compound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide sequences of the present invention.

Table 2 shows the GenBank identification number and annotation of thenearest GenBank homolog for polypeptides of the invention. Theprobability score for the match between each polypeptide and its GenBankhomolog is also shown.

Table 3 shows structural features of polypeptide sequences of theinvention, including predicted motifs and domains, along with themethods, algorithms, and searchable databases used for analysis of thepolypeptides.

Table 4 lists the cDNA and genomic DNA fragments which were used toassemble polynucleotide sequences of the invention, along with selectedfragments of the polynucleotide sequences. Table 5 shows therepresentative cDNA library for polynucleotides of the invention.

Table 6 provides an appendix which describes the tissues and vectorsused for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyze thepolynucleotides and polypeptides of the invention, along with applicabledescriptions, references, and threshold parameters.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular machines, materials and methods described, as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Definitions

“TRICH” refers to the amino acid sequences of substantially purifiedTRICH obtained from any species, particularly a mammalian species,including bovine, ovine, porcine, murine, equine, and human, and fromany source, whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which intensifies or mimics thebiological activity of TRICH. Agonists may include proteins, nucleicacids, carbohydrates, small molecules, or any other compound orcomposition which modulates the activity of TRICH either by directlyinteracting with TRICH or by acting on components of the biologicalpathway in which TRICH participates.

An “allelic variant” is an alternative form of the gene encoding TRICH.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. A gene may have none,one, or many allelic variants of its naturally occurring form. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding TRICH include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polypeptide the same as TRICH or a polypeptide with atleast one functional characteristic of TRICH. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingTRICH, and improper or unexpected hybridization to allelic variants,with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding TRICH. The encoded protein may also be“altered,” and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent TRICH. Deliberate amino acid substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of TRICHis retained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, and positively charged amino acids mayinclude lysine and arginine. Amino acids with uncharged polar sidechains having similar hydrophilicity values may include: asparagine andglutamine; and serine and threonine. Amino acids with uncharged sidechains having similar hydrophilicity values may include: leucine,isoleucine, and valine; glycine and alanine; and phenylalanine andtyrosine.

The terms “amino acid” and “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules.Where “amino acid sequence” is recited to refer to a sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

“Amplification” relates to the production of additional copies of anucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

The term “antagonist” refers to a molecule which inhibits or attenuatesthe biological activity of TRICH. Antagonists may include proteins suchas antibodies, nucleic acids, carbohydrates, small molecules, or anyother compound or composition which modulates the activity of TRICHeither by directly interacting with TRICH or by acting on components ofthe biological pathway in which TRICH participates.

The term “antibody” refers to intact immunoglobulin molecules as well asto fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding an epitopic determinant. Antibodies that bind TRICHpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant” refers to that region of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (particular regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “antisense” refers to any composition capable of base-pairingwith the “sense” (coding) strand of a specific nucleic acid sequence.Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA);oligonucleotides having modified backbone linkages such asphosphorothioates, methylphosphonates, or benzylphosphonates;oligonucleotides having modified sugar groups such as 2′-methoxyethylsugars or 2′-methoxyethoxy sugars; or oligonucleotides having modifiedbases such as 5-methyl cytosine, 2′-deoxyuracil, or7-deaza-2′-deoxyguanosine. Antisense molecules may be produced by anymethod including chemical synthesis or transcription. Once introducedinto a cell, the complementary antisense molecule base-pairs with anaturally occurring nucleic acid sequence produced by the cell to formduplexes which block either transcription or translation. Thedesignation “negative” or “minus” can refer to the antisense strand, andthe designation “positive” or “plus” can refer to the sense strand of areference DNA molecule.

The term “biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic TRICH, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

“Complementary” describes the relationship between two single-strandednucleic acid sequences that anneal by base-pairing. For example,5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

A “composition comprising a given polynucleotide sequence” and a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encodingTRICH or fragments of TRICH may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beensubjected to repeated DNA sequence analysis to resolve uncalled bases,extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.)in the 5′ and/or the 3′ direction, and resequenced, or which has beenassembled from one or more overlapping cDNA, EST, or genomic DNAfragments using a computer program for fragment assembly, such as theGELVIEW fragment assembly system (GCG, Madison Wis.) or Phrap(University of Washington, Seattle Wash.). Some sequences have been bothextended and assembled to produce the consensus sequence.

“Conservative amino acid substitutions” are those substitutions that arepredicted to least interfere with the properties of the originalprotein, i.e., the structure and especially the function of the proteinis conserved and not significantly changed by such substitutions. Thetable below shows amino acids which may be substituted for an originalamino acid in a protein and which are regarded as conservative aminoacid substitutions. Original Residue Conservative Substitution Ala Gly,Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn,Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, ValLeu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, TyrSer Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu,Thr

Conservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to a chemically modified polynucleotide orpolypeptide. Chemical modifications of a polynucleotide can include, forexample, replacement of hydrogen by an alkyl, acyl, hydroxyl, or aminogroup. A derivative polynucleotide encodes a polypeptide which retainsat least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

A “detectable label” refers to a reporter molecule or enzyme that iscapable of generating a measurable signal and is covalently ornoncovalently joined to a polynucleotide or polypeptide.

A “fragment” is a unique portion of TRICH or the polynucleotide encodingTRICH which is identical in sequence to but shorter in length than theparent sequence. A fragment may comprise up to the entire length of thedefined sequence, minus one nucleotide/amino acid residue. For example,a fragment may comprise from 5 to 1000 contiguous nucleotides or aminoacid residues. A fragment used as a probe, primer, antigen, therapeuticmolecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25,30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotidesor amino acid residues in length. Fragments may be preferentiallyselected from certain regions of a molecule. For example, a polypeptidefragment may comprise a certain length of contiguous amino acidsselected from the first 250 or 500 amino acids (or first 25% or 50%) ofa polypeptide as shown in a certain defined sequence. Clearly theselengths are exemplary, and any length that is supported by thespecification, including the Sequence Listing, tables, and figures, maybe encompassed by the present embodiments.

A fragment of SEQ ID NO:14-26 comprises a region of uniquepolynucleotide sequence that specifically identifies SEQ ID NO:14-26,for example, as distinct from any other sequence in the genome fromwhich the fragment was obtained. A fragment of SEQ ID NO:14-26 isuseful, for example, in hybridization and amplification technologies andin analogous methods that distinguish SEQ ID NO:14-26 from relatedpolynucleotide sequences. The precise length of a fragment of SEQ IDNO:14-26 and the region of SEQ ID NO:14-26 to which the fragmentcorresponds are routinely determinable by one of ordinary skill in theart based on the intended purpose for the fragment.

A fragment of SEQ ID NO:1-13 is encoded by a fragment of SEQ IDNO:14-26. A fragment of SEQ ID NO:1-13 comprises a region of uniqueamino acid sequence that specifically identifies SEQ ID NO:1-13. Forexample, a fragment of SEQ ID NO:1-13 is useful as an immunogenicpeptide for the development of antibodies that specifically recognizeSEQ ID NO:1-13. The precise length of a fragment of SEQ ID NO:1-13 andthe region of SEQ ID NO:1-13 to which the fragment corresponds areroutinely determinable by one of ordinary skill in the art based on theintended purpose for the fragment.

A “full length” polynucleotide sequence is one containing at least atranslation initiation codon (e.g., methionine) followed by an openreading frame and a translation termination codon. A “full length”polynucleotide sequence encodes a “full length” polypeptide sequence.

“Homology” refers to sequence similarity or, interchangeably, sequenceidentity, between two or more polynucleotide sequences or two or morepolypeptide sequences.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determinedusing the default parameters of the CLUSTAL V algorithm as incorporatedinto the MEGALIGN version 3.12e sequence alignment program. This programis part of the LASERGENE software package, a suite of molecularbiological analysis programs (DNASTAR, Madison Wis.). CLUSTAL V isdescribed in Higgins, D. G. and P. M. Sharp (1989) CABIOS 5:151-153 andin Higgins, D. G. et al. (1992) CABIOS 8:189-191. For pairwisealignments of polynucleotide sequences, the default parameters are setas follows: Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4.The “weighted” residue weight table is selected as the default. Percentidentity is reported by CLUSTAL V as the “percent similarity” betweenaligned polynucleotide sequences.

Alternatively, a suite of commonly used and freely available sequencecomparison algorithms is provided by the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), whichis available from several sources, including the NCBI Bethesda, Md., andon the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLASTsoftware suite includes various sequence analysis programs including“blastn,” that is used to align a known polynucleotide sequence withother polynucleotide sequences from a variety of databases. Alsoavailable is a tool called “BLAST 2 Sequences” that is used for directpairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” canbe accessed and used interactively athttp://www.ncbi.nlm.nih.gov/gorf/bl2.html. The “BLAST 2 Sequences” toolcan be used for both blastn and blastp (discussed below). BLAST programsare commonly used with gap and other parameters set to default settings.For example, to compare two nucleotide sequences, one may use blastnwith the “BLAST 2 Sequences” tool Version 2.0.12 (Apr.-21-2000) set atdefault parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: −2

Open Gap: 5 and Extension Gap: 2 penalties

Gap×drop-off: 50

Expect: 10

Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire definedsequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of afragment taken from a larger, defined sequence, for instance, a fragmentof at least 20, at least 30, at least 40, at least 50, at least 70, atleast 100, or at least 200 contiguous nucleotides. Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in the tables, figures, or SequenceListing, may be used to describe a length over which percentage identitymay be measured.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code. It is understood that changes in a nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide.

Percent identity between polypeptide sequences may be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program (described andreferenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table. As with polynucleotidealignments, the percent identity is reported by CLUSTAL V as the“percent similarity” between aligned polypeptide sequence pairs.

Alternatively the NCBI BLAST software suite may be used. For example,for a pairwise comparison of two polypeptide sequences, one may use the“BLAST 2 Sequences” tool Version 2.0.12 (Apr.-21-2000) with blastp setat default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Open Gap: 11 and Extension Gap: 1 penalties

Gap×drop-off: 50

Expect: 10

Word Size: 3

Filter: on

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size and whichcontain all of the elements required for chromosome replication,segregation and maintenance.

The term “humanized antibody” refers to an antibody molecule in whichthe amino acid sequence in the non-antigen binding regions has beenaltered so that the antibody more closely resembles a human antibody,and still retains its original binding ability.

“Hybridization” refers to the process by which a polynucleotide strandanneals with a complementary strand through base pairing under definedhybridization conditions. Specific hybridization is an indication thattwo nucleic acid sequences share a high degree of complementarity.Specific hybridization complexes form under permissive annealingconditions and remain hybridized after the “washing” step(s). Thewashing step(s) is particularly important in determining the stringencyof the hybridization process, with more stringent conditions allowingless non-specific binding, i.e., binding between pairs of nucleic acidstrands that are not perfectly matched. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may be consistent among hybridizationexperiments, whereas wash conditions may be varied among experiments toachieve the desired stringency, and therefore hybridization specificity.Permissive annealing conditions occur, for example, at 68° C. in thepresence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/mlsheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, withreference to the temperature under which the wash step is carried out.Such wash temperatures are typically selected to be about 5° C. to 20°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An equation forcalculating T_(m) and conditions for nucleic acid hybridization are wellknown and can be found in Sambrook, J. et al. (1989) Molecular Cloning:A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; specifically see volume 2, chapter 9.

High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68° C. in the presenceof about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively,temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSCconcentration may be varied from about 0.1 to 2×SSC, with SDS beingpresent at about 0.1%. Typically, blocking reagents are used to blocknon-specific hybridization. Such blocking reagents include, forinstance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml.Organic solvent, such as formamide at a concentration of about 35-50%v/v, may also be used under particular circumstances, such as forRNA:DNA hybridizations. Useful variations on these wash conditions willbe readily apparent to those of ordinary skill in the art.Hybridization, particularly under high stringency conditions, may besuggestive of evolutionary similarity between the nucleotides. Suchsimilarity is strongly indicative of a similar role for the nucleotidesand their encoded polypeptides.

The term “hybridization complex” refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary bases. A hybridization complex may be formed insolution (e.g., C₀t or R₀t analysis) or formed between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., paper, membranes, filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed).

The words “insertion” and “addition” refer to changes in an amino acidor nucleotide sequence resulting in the addition of one or more aminoacid residues or nucleotides, respectively.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment ofTRICH which is capable of eliciting an immune response when introducedinto a living organism, for example, a mammal. The term “immunogenicfragment” also includes any polypeptide or oligopeptide fragment ofTRICH which is useful in any of the antibody production methodsdisclosed herein or known in the art.

The term “microarray” refers to an arrangement of a plurality ofpolynucleotides, polypeptides, or other chemical compounds on asubstrate.

The terms “element” and “array element” refer to a polynucleotide,polypeptide, or other chemical compound having a unique and definedposition on a microarray.

The term “modulate” refers to a change in the activity of TRICH. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of TRICH.

The phrases “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material.

“Operably linked” refers to the situation in which a first nucleic acidsequence is placed in a functional relationship with a second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Operably linked DNA sequences may be in close proximityor contiguous and, where necessary to join two protein coding regions,in the same reading frame.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

“Post-translational modification” of an TRICH may involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and other modifications known in the art. These processes mayoccur synthetically or biochemically. Biochemical modifications willvary by cell type depending on the enzymatic milieu of TRICH.

“Probe” refers to nucleic acid sequences encoding TRICH, theircomplements, or fragments thereof, which are used to detect identical,allelic or related nucleic acid sequences. Probes are isolatedoligonucleotides or polynucleotides attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes. “Primers” are short nucleic acids,usually DNA oligonucleotides, which may be annealed to a targetpolynucleotide by complementary base-pairing. The primer may then beextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification (and identification) of a nucleicacid sequence, e.g., by the polymerase chain reaction (PCR).

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 20, 25, 30, 40, 50,60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of thedisclosed nucleic acid sequences. Probes and primers may be considerablylonger than these examples, and it is understood that any lengthsupported by the specification, including the tables, figures, andSequence Listing, may be used.

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook, J. et al. (1989) Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; Ausubel, F. M. et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New YorkN.Y.; Innis, M. et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, San Diego Calif. PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers are selected using software known inthe art for such purpose. For example, OLIGO 4.06 software is useful forthe selection of PCR primer pairs of up to 100 nucleotides each, and forthe analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope. The Primer3 primerselection program (available to the public from the WhiteheadInstitute/MIT Center for Genome Research, Cambridge Mass.) allows theuser to input a “mispriming library,” in which sequences to avoid asprimer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Center, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

A “recombinant nucleic acid” is a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook, supra. The term recombinant includes nucleic acids thathave been altered solely by addition, substitution, or deletion of aportion of the nucleic acid. Frequently, a recombinant nucleic acid mayinclude a nucleic acid sequence operably linked to a promoter sequence.Such a recombinant nucleic acid may be part of a vector that is used,for example, to transform a cell.

Alternatively, such recombinant nucleic acids may be part of a viralvector, e.g., based on a vaccinia virus, that could be use to vaccinatea mammal wherein the recombinant nucleic acid is expressed, inducing aprotective immunological response in the mammal.

A “regulatory element” refers to a nucleic acid sequence usually derivedfrom untranslated regions of a gene and includes enhancers, promoters,introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elementsinteract with host or viral proteins which control transcription,translation, or RNA stability.

“Reporter molecules” are chemical or biochemical moieties used forlabeling a nucleic acid, amino acid, or antibody. Reporter moleculesinclude radionuclides; enzymes; fluorescent, chemiluminescent, orchromogenic agents; substrates; cofactors; inhibitors; magneticparticles; and other moieties known in the art.

An “RNA equivalent,” in reference to a DNA sequence, is composed of thesame linear sequence of nucleotides as the reference DNA sequence withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining TRICH, nucleic acids encoding TRICH, or fragments thereof maycomprise a bodily fluid; an extract from a cell, chromosome, organelle,or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, insolution or bound to a substrate; a tissue; a tissue print; etc.

The terms “specific binding” and “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, anantagonist, a small molecule, or any natural or synthetic bindingcomposition. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide comprisingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least 60% free, preferably at least75% free, and most preferably at least 90% free from other componentswith which they are naturally associated.

A “substitution” refers to the replacement of one or more amino acidresidues or nucleotides by different amino acid residues or nucleotides,respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

A “transcript image” refers to the collective pattern of gene expressionby a particular cell type or tissue under given conditions at a giventime.

“Transformation” describes a process by which exogenous DNA isintroduced into a recipient cell. Transformation may occur under naturalor artificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, bacteriophageor viral infection, electroporation, heat shock, lipofection, andparticle bombardment. The term “transformed cells” includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “transgenic organism,” as used herein, is any organism, including butnot limited to animals and plants, in which one or more of the cells ofthe organism contains heterologous nucleic acid introduced by way ofhuman intervention, such as by transgenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.The transgenic organisms contemplated in accordance with the presentinvention include bacteria, cyanobacteria, fungi, plants and animals.The isolated DNA of the present invention can be introduced into thehost by methods known in the art, for example infection, transfection,transformation or transconjugation. Techniques for transferring the DNAof the present invention into such organisms are widely known andprovided in references such as Sambrook et al. (1989), supra.

A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 40% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May-07-1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% or greater sequence identityover a certain defined length. A variant may be described as, forexample, an “allelic” (as defined above), “splice,” “species,” or“polymorphic” variant. A splice variant may have significant identity toa reference molecule, but will generally have a greater or lesser numberof polynucleotides due to alternative splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or lack domains that are present in the referencemolecule. Species variants are polynucleotide sequences that vary fromone species to another. The resulting polypeptides will generally havesignificant amino acid identity relative to each other. A polymorphicvariant is a variation in the polynucleotide sequence of a particulargene between individuals of a given species. Polymorphic variants alsomay encompass “single nucleotide polymorphisms” (SNPs) in which thepolynucleotide sequence varies by one nucleotide base. The presence ofSNPs may be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” toolVersion 2.0.9 (May-07-1999) set at default parameters. Such a pair ofpolypeptides may show, for example, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% or greater sequence identity over a certain definedlength of one of the polypeptides.

The Invention

The invention is based on the discovery of new human transporters andion channels (TRICH), the polynucleotides encoding TRICH, and the use ofthese compositions for the diagnosis, treatment, or prevention oftransport, neurological, muscle, and immunological disorders.

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide sequences of the invention. Each polynucleotide and itscorresponding polypeptide are correlated to a single Incyte projectidentification number (Incyte Project ID). Each polypeptide sequence isdenoted by both a polypeptide sequence identification number(Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number(Incyte Polypeptide ID) as shown. Each polynucleotide sequence isdenoted by both a polynucleotide sequence identification number(Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensussequence number (Incyte Polynucleotide ID) as shown.

Table 2 shows sequences with homology to the polypeptides of theinvention as identified by BLAST analysis against the GenBank protein(genpept) database. Columns 1 and 2 show the polypeptide sequenceidentification number (Polypeptide SEQ ID NO:) and the correspondingIncyte polypeptide sequence number (Incyte Polypeptide ID) forpolypeptides of the invention. Column 3 shows the GenBank identificationnumber (Genbank ID NO:) of the nearest GenBank homolog. Column 4 showsthe probability score for the match between each polypeptide and itsGenBank homolog. Column 5 shows the annotation of the GenBank homologalong with relevant citations where applicable, all of which areexpressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of theinvention. Columns 1 and 2 show the polypeptide sequence identificationnumber (SEQ ID NO:) and the corresponding Incyte polypeptide sequencenumber (Incyte Polypeptide ID) for each polypeptide of the invention.Column 3 shows the number of amino acid residues in each polypeptide.Column 4 shows potential phosphorylation sites, and column 5 showspotential glycosylation sites, as determined by the MOTIFS program ofthe GCG sequence analysis software package (Genetics Computer Group,Madison Wis.). Column 6 shows amino acid residues comprising signaturesequences, domains, and motifs. Column 7 shows analytical methods forprotein structure/function analysis and in some cases, searchabledatabases to which the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of theinvention, and these properties establish that the claimed polypeptidesare transporters and ion channels. For example, SEQ ID NO:12 is 95%identical, from residue M1 to residue T669, to human amiloride sensitivesodium channel delta subunit (GenBank ID g1066457) as determined by theBasic Local Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 0.0, which indicates the probability of obtainingthe observed polypeptide sequence alignment by chance. SEQ ID NO:12 alsocontains an amiloride-sensitive sodium channel domain as determined bysearching for statistically significant matches in the hidden Markovmodel (HMM)-based PFAM database of conserved protein family domains.(See Table 3.) Data from BLIMPS analyses provide further corroborativeevidence that SEQ ID NO:12 is an amiloride-sensitive sodium channel. SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3 ,SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,and SEQ ID NO:13 were analyzed and annotated in a similar manner. Thealgorithms and parameters for the analysis of SEQ ID NO:1-13 aredescribed in Table 7.

As shown in Table 4, the full length polynucleotide sequences of thepresent invention were assembled using cDNA sequences or coding (exon)sequences derived from genomic DNA, or any combination of these twotypes of sequences. Columns 1 and 2 list the polynucleotide sequenceidentification number (Polynucleotide SEQ ID NO:) and the correspondingIncyte polynucleotide consensus sequence number (Incyte PolynucleotideID) for each polynucleotide of the invention. Column 3 shows the lengthof each polynucleotide sequence in basepairs. Column 4 lists fragmentsof the polynucleotide sequences which are useful, for example, inhybridization or amplification technologies that identify SEQ IDNO:14-26 or that distinguish between SEQ ID NO:14-26 and relatedpolynucleotide sequences. Column 5 shows identification numberscorresponding to cDNA sequences, coding sequences (exons) predicted fromgenomic DNA, and/or sequence assemblages comprised of both cDNA andgenomic DNA. These sequences were used to assemble the full lengthpolynucleotide sequences of the invention. Columns 6 and 7 of Table 4show the nucleotide start (5′) and stop (3′) positions of the cDNA andgenomic sequences in column 5 relative to their respective full lengthsequences.

The identification numbers in Column 5 of Table 4 may referspecifically, for example, to Incyte cDNAs along with theircorresponding cDNA libraries. 1581463F6 is the identification number ofan Incyte cDNA sequence, and DUODNOT01 is the cDNA library from which itis derived. Incyte cDNAs for which cDNA libraries are not indicated werederived from pooled cDNA libraries (e.g., 70558367V1). Alternatively,the identification numbers in column 5 may refer to GenBank cDNAs orESTs which contributed to the assembly of the full length polynucleotidesequences. Alternatively, the identification numbers in column 5 mayrefer to coding regions predicted by Genscan analysis of genomic DNA.For example, GNN.g6624821_(—)028 is the identification number of aGenscan-predicted coding sequence, with g6624821 being the GenBankidentification number of the sequence to which Genscan was applied. TheGenscan-predicted coding sequences may have been edited prior toassembly. (See Example IV.) Alternatively, the identification numbers incolumn 5 may refer to assemblages of both cDNA and Genscan-predictedexons brought together by an “exon stitching” algorithm. For example,FL1621218_(—)00001 represents a “stitched” sequence in which 1621218 isthe identification number of the cluster of sequences to which thealgorithm was applied, and 00001 is the number of the predictiongenerated by the algorithm. (See Example V.) Alternatively, theidentification numbers in column 5 may refer to assemblages of both cDNAand Genscan-predicted exons brought together by an “exon-stretching”algorithm. (See Example V.) In some cases, Incyte cDNA coverageredundant with the sequence coverage shown in column 5 was obtained toconfirm the final consensus polynucleotide sequence, but the relevantIncyte cDNA identification numbers are not shown.

Table 5 shows the representative cDNA libraries for those full lengthpolynucleotide sequences which were assembled using Incyte cDNAsequences. The representative cDNA library is the Incyte cDNA librarywhich is most frequently represented by the Incyte cDNA sequences whichwere used to assemble and confirm the above polynucleotide sequences.The tissues and vectors which were used to construct the cDNA librariesshown in Table 5 are described in Table 6.

The invention also encompasses TRICH variants. A preferred TRICH variantis one which has at least about 80%, or alternatively at least about90%, or even at least about 95% amino acid sequence identity to theTRICH amino acid sequence, and which contains at least one functional orstructural characteristic of TRICH.

The invention also encompasses polynucleotides which encode TRICH. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:14-26, which encodes TRICH. The polynucleotide sequences of SEQ IDNO:14-26, as presented in the Sequence Listing, embrace the equivalentRNA sequences, wherein occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The invention also encompasses a variant of a polynucleotide sequenceencoding TRICH. In particular, such a variant polynucleotide sequencewill have at least about 70%, or alternatively at least about 85%, oreven at least about 95% polynucleotide sequence identity to thepolynucleotide sequence encoding TRICH. A particular aspect of theinvention encompasses a variant of a polynucleotide sequence comprisinga sequence selected from the group consisting of SEQ ID NO:14-26 whichhas at least about 70%, or alternatively at least about 85%, or even atleast about 95% polynucleotide sequence identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO:14-26. Any oneof the polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of TRICH.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding TRICH, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring TRICH, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode TRICH and its variants aregenerally capable of hybridizing to the nucleotide sequence of thenaturally occurring TRICH under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding TRICH or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding TRICH and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeTRICH and TRICH derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding TRICH or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:14-26 and fragments thereofunder various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.) Hybridization conditions, includingannealing and wash conditions, are described in “Definitions.”

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase 1 , SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems),thermostable T7 polymerase (Amersham Pharmacia Biotech, PiscatawayN.J.), or combinations of polymerases and proofreading exonucleases suchas those found in the ELONGASE amplification system (Life Technologies,Gaithersburg Md.). Preferably, sequence preparation is automated withmachines such as the MICROLAB 2200 liquid transfer system (Hamilton,Reno Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABICATALYST 800 thermal cycler (Applied Biosystems). Sequencing is thencarried out using either the ABI 373 or 377 DNA sequencing system(Applied Biosystems), the MEGABACE 1000 DNA sequencing system (MolecularDynamics, Sunnyvale Calif.), or other systems known in the art. Theresulting sequences are analyzed using a variety of algorithms which arewell known in the art. (See, e.g., Ausubel, F. M. (1997) Short Protocolsin Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7;Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley V C H,New York N.Y., pp. 856-853.)

The nucleic acid sequences encoding TRICH may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppliedBiosystems), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode TRICH may be cloned in recombinant DNAmolecules that direct expression of TRICH, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express TRICH.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterTRICH-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

The nucleotides of the present invention may be subjected to DNAshuffling techniques such as MOLECULARBREEDING (Maxygen Inc., SantaClara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al.(1999) Nat. Biotechnol. 17:793-797; Christians, F .C. et al. (1999) Nat.Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.14:315-319) to alter or improve the biological properties of TRICH, suchas its biological or enzymatic activity or its ability to bind to othermolecules or compounds. DNA shuffling is a process by which a library ofgene variants is produced using PCR-mediated recombination of genefragments. The library is then subjected to selection or screeningprocedures that identify those gene variants with the desiredproperties. These preferred variants may then be pooled and furthersubjected to recursive rounds of DNA shuffling and selection/screening.Thus, genetic diversity is created through “artificial” breeding andrapid molecular evolution. For example, fragments of a single genecontaining random point mutations may be recombined, screened, and thenreshuffled until the desired properties are optimized. Alternatively,fragments of a given gene may be recombined with fragments of homologousgenes in the same gene family, either from the same or differentspecies, thereby maximizing the genetic diversity of multiple naturallyoccurring genes in a directed and controllable manner.

In another embodiment, sequences encoding TRICH may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223;and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.)Alternatively, TRICH itself or a fragment thereof may be synthesizedusing chemical methods. For example, peptide synthesis can be performedusing various solution-phase or solid-phase techniques. (See, e.g.,Creighton, T. (1984) Proteins, Structures and Molecular Properties, W HFreeman, New York N.Y., pp. 55-60; and Roberge, J. Y. et al. (1995)Science 269:202-204.) Automated synthesis may be achieved using the ABI431A peptide synthesizer (Applied Biosystems). Additionally, the aminoacid sequence of TRICH, or any part thereof, may be altered duringdirect synthesis and/or combined with sequences from other proteins, orany part thereof, to produce a variant polypeptide or a polypeptidehaving a sequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g., Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, supra, pp. 28-53.)

In order to express a biologically active TRICH, the nucleotidesequences encoding TRICH or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding TRICH. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding TRICH. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding TRICH and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding TRICH andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding TRICH. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See,e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; TheMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad.Sci. USA 81:3655-3659; and Harrington, J. J. et al. (1997) Nat. Genet.15:345-355.) Expression vectors derived from retroviruses, adenoviruses,or herpes or vaccinia viruses, or from various bacterial plasmids, maybe used for delivery of nucleotide sequences to the targeted organ,tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998)Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad.Sci. USA 90(13):6340-6344; Buller, R. M. et al. (1985) Nature317(6040):813-815; McGregor, D. P. et al. (1994) Mol. Immunol.31(3):219-226; and Verma, I. M. and N. Somia (1997) Nature 389:239-242.)The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding TRICH. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding TRICH can be achievedusing a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene,La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Ligation ofsequences encoding TRICH into the vector's multiple cloning sitedisrupts the lacZ gene, allowing a colorimetric screening procedure foridentification of transformed bacteria containing recombinant molecules.In addition, these vectors may be useful for in vitro transcription,dideoxy sequencing, single strand rescue with helper phage, and creationof nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of TRICH are needed, e.g. for the production of antibodies,vectors which direct high level expression of TRICH may be used. Forexample, vectors containing the strong, inducible SP6 or T7bacteriophage promoter may be used.

Yeast expression systems may be used for production of TRICH. A numberof vectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH promoters, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign sequences into the hostgenome for stable propagation. (See, e.g., Ausubel, 1995, supra; Bitter,G. A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C. A. etal. (1994) Bio/Technology 12:181-184.)

Plant systems may also be used for expression of TRICH. Transcription ofsequences encoding TRICH may be driven by viral promoters, e.g., the 35Sand 19S promoters of CaMV used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., The McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New YorkN.Y., pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding TRICH may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses TRICH in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. etal. (1997) Nat. Genet. 15:345-355.)

For long term production of recombinant proteins in mammalian systems,stable expression of TRICH in cell lines is preferred. For example,sequences encoding TRICH can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk′ and apr′ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG-418; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin,F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable geneshave been described, e.g., trpB and hisD, which alter cellularrequirements for metabolites. (See, e.g., Hartman, S. C. and R. C.Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051.) Visiblemarkers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),β glucuronidase and its substrate β-glucuronide, or luciferase and itssubstrate luciferin may be used. These markers can be used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system.(See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingTRICH is inserted within a marker gene sequence, transformed cellscontaining sequences encoding TRICH can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding TRICH under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingTRICH and that express TRICH may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression ofTRICH using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on TRICH is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St. Paul Minn., Sect. IV; Coligan, J. E.et al. (1997) Current Protocols in Immunology, Greene Pub. Associatesand Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998)Immunochemical Protocols, Humana Press, Totowa N.J.)

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding TRICH includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding TRICH,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamPharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitablereporter molecules or labels which may be used for ease of detectioninclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding TRICH may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeTRICH may be designed to contain signal sequences which direct secretionof TRICH through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” or “pro” form ofthe protein may also be used to specify protein targeting, folding,and/or activity. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available fromthe American Type Culture Collection (ATCC, Manassas Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding TRICH may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric TRICHprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of, TRICH activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the TRICH encodingsequence and the heterologous protein sequence, so that TRICH may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel (1995, supra, ch. 10). A variety of commercially available kitsmay also be used to facilitate expression and purification of fusionproteins.

In a further embodiment of the invention, synthesis of radiolabeledTRICH may be achieved in vitro using the TNT rabbit reticulocyte lysateor wheat germ extract system (Promega). These systems coupletranscription and translation of protein-coding sequences operablyassociated with the T7, T3, or SP6 promoters. Translation takes place inthe presence of a radiolabeled amino acid precursor, for example,³⁵S-methionine.

TRICH of the present invention or fragments thereof may be used toscreen for compounds that specifically bind to TRICH. At least one andup to a plurality of test compounds may be screened for specific bindingto TRICH. Examples of test compounds include antibodies,oligonucleotides, proteins (e.g., receptors), or small molecules.

In one embodiment, the compound thus identified is closely related tothe natural ligand of TRICH, e.g., a ligand or fragment thereof, anatural substrate, a structural or functional mimetic, or a naturalbinding partner. (See, e.g., Coligan, J. E. et al. (1991) CurrentProtocols in Immunology 1(2): Chapter 5.) Similarly, the compound can beclosely related to the natural receptor to which TRICH binds, or to atleast a fragment of the receptor, e.g., the ligand binding site. Ineither case, the compound can be rationally designed using knowntechniques. In one embodiment, screening for these compounds involvesproducing appropriate cells which express TRICH, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing TRICH or cellmembrane fractions which contain TRICH are then contacted with a testcompound and binding, stimulation, or inhibition of activity of eitherTRICH or the compound is analyzed.

An assay may simply test binding of a test compound to the polypeptide,wherein binding is detected by a fluorophore, radioisotope, enzymeconjugate, or other detectable label. For example, the assay maycomprise the steps of combining at least one test compound with TRICH,either in solution or affixed to a solid support, and detecting thebinding of TRICH to the compound. Alternatively, the assay may detect ormeasure binding of a test compound in the presence of a labeledcompetitor. Additionally, the assay may be carried out using cell-freepreparations, chemical libraries, or natural product mixtures, and thetest compound(s) may be free in solution or affixed to a solid support.

TRICH of the present invention or fragments thereof may be used toscreen for compounds that modulate the activity of TRICH. Such compoundsmay include agonists, antagonists, or partial or inverse agonists. Inone embodiment, an assay is performed under conditions permissive for

In another embodiment, polynucleotides encoding TRICH or their mammalianhomologs may be “knocked out” in an animal model system using homologousrecombination in embryonic stem (ES) cells. Such techniques are wellknown in the art and are useful for the generation of animal models ofhuman disease. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No.5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cellline, are derived from the early mouse embryo and grown in culture. TheES cells are transformed with a vector containing the gene of interestdisrupted by a marker gene, e.g., the neomycin phosphotransferase gene(neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vectorintegrates into the corresponding region of the host genome byhomologous recombination. Alternatively, homologous recombination takesplace using the Cre-loxP system to knockout a gene of interest in atissue- or developmental stage-specific manner (Marth, J. D. (1996)Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic AcidsRes. 25:4323-4330). Transformed ES cells are identified andmicroinjected into mouse cell blastocysts such as those from the C57BL/6mouse strain. The blastocysts are surgically transferred topseudopregnant dams, and the resulting chimeric progeny are genotypedand bred to produce heterozygous or homozygous strains. Transgenicanimals thus generated may be tested with potential therapeutic or toxicagents.

Polynucleotides encoding TRICH may also be manipulated in vitro in EScells derived from human blastocysts. Human ES cells have the potentialto differentiate into at least eight separate cell lineages includingendoderm, mesoderm, and ectodermal cell types. These cell lineagesdifferentiate into, for example, neural cells, hematopoietic lineages,and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding TRICH can also be used to create “knockin”humanized animals (pigs) or transgenic animals (mice or rats) to modelhuman disease. With knockin technology, a region of a polynucleotideencoding TRICH is injected into animal ES cells, and the injectedsequence integrates into the animal cell genome. Transformed cells areinjected into blastulae, and the blastulae are implanted as describedabove. Transgenic progeny or inbred lines are studied and treated withpotential pharmaceutical agents to obtain information on treatment of ahuman disease. Alternatively, a mammal inbred to overexpress TRICH,e.g., by secreting TRICH in its milk, may also serve as a convenientsource of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev.4:55-74).

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of TRICH and transporters and ionchannels. In addition, the expression of TRICH is closely associatedwith fetal tissues and neoplasms associated with tissues of epidermalorigin, and with rapidly dividing cells. Therefore, TRICH appears toplay a role in transport, neurological, muscle, and immunologicaldisorders. In the treatment of disorders associated with increased TRICHexpression or activity, it is desirable to decrease the expression oractivity of TRICH. In the treatment of disorders associated withdecreased TRICH expression or activity, it is desirable to increase theexpression or activity of TRICH.

Therefore, in one embodiment, TRICH or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of TRICH. Examples ofsuch disorders include, but are not limited to, a transport disordersuch as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia,cystic fibrosis, Becker's muscular dystrophy, Bell's palsy,Charcot-Marie Tooth disease, diabetes mellitus, diabetes insipidus,diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodicparalysis, normokalemic periodic paralysis, Parkinson's disease,malignant hyperthermia, multidrug resistance, myasthenia gravis,myotonic dystrophy, catatonia, tardive dyskinesia, dystonias, peripheralneuropathy, cerebral neoplasms, prostate cancer, cardiac disordersassociated with transport, e.g., angina, bradyarrythmia, tachyarrythmia,hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nemalinemyopathy, centronuclear myopathy, lipid myopathy, mitochondrialmyopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis,inclusion body myositis, infectious myositis, polymyositis, neurologicaldisorders associated with transport, e.g., Alzheimer's disease, amnesia,bipolar disorder, dementia, depression, epilepsy, Tourette's disorder,paranoid psychoses, and schizophrenia, and other disorders associatedwith transport, e.g., neurofibromatosis, postherpetic neuralgia,trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson'sdisease, cataracts, infertility, pulmonary artery stenosis,sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave'sdisease, goiter, Cushing's disease, Addison's disease, glucose-galactosemalabsorption syndrome, hypercholesterolemia, adrenoleukodystrophy,Zellweger syndrome, Menkes disease, occipital horn syndrome, von Gierkedisease, cystinuria, iminoglycinuria, Hartup disease, and Fanconidisease; a neurological disorder such as epilepsy, ischemiccerebrovascular disease, stroke, cerebral neoplasms, Alzheimer'sdisease, Pick's disease, Huntington's disease, dementia, Parkinson'sdisease and other extrapyramidal disorders, amyotrophic lateralsclerosis and other motor neuron disorders, progressive neural muscularatrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosisand other demyelinating diseases, bacterial and viral meningitis, brainabscess, subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease, prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; a muscle disorder such ascardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker'smuscular dystrophy, myotonic dystrophy, central core disease, nemalinemyopathy, centronuclear myopathy, lipid myopathy, mitochondrialmyopathy, infectious myositis, polymyositis, dermatomyositis, inclusionbody myositis, thyrotoxic myopathy, ethanol myopathy, angina,anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing'ssyndrome, hypertension, hypoglycemia, myocardial infarction, migraine,pheochromocytoma, and myopathies including encephalopathy, epilepsy,Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder,ophthalmoplegia, and acid maltase deficiency (AMD, also known as Pompe'sdisease); and an immunological disorder such as acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermaldystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma.

In another embodiment, a vector capable of expressing TRICH or afragment or derivative thereof may be administered to a subject to treator prevent a disorder associated with decreased expression or activityof TRICH including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantiallypurified TRICH in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associatedwith decreased expression or activity of TRICH including, but notlimited to, those provided above.

In still another embodiment, an agonist which modulates the activity ofTRICH may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of TRICH including, butnot limited to, those listed above.

In a further embodiment, an antagonist of TRICH may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of TRICH. Examples of such disorders include, butare not limited to, those transport, neurological, muscle, andimmunological disorders described above. In one aspect, an antibodywhich specifically binds TRICH may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissues which express TRICH.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding TRICH may be administered to a subject to treator prevent a disorder associated with increased expression or activityof TRICH including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of TRICH may be produced using methods which are generallyknown in the art. In particular, purified TRICH may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind TRICH. Antibodies to TRICH may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are generally preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith TRICH or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to TRICH have an amino acid sequence consisting of atleast about 5 amino acids, and generally will consist of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein. Short stretches of TRICH amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to TRICH may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S. P. etal. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce TRICH-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for TRICH mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)₂ fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between TRICH and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering TRICH epitopes is generally used, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for TRICH. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of TRICH-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple TRICH epitopes, represents the average affinity,or avidity, of the antibodies for TRICH. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular TRICH epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theTRICH-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of TRICH, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I: APractical Approach, IRL Press, Washington D.C.; Liddell, J. E. and A.Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley &Sons, New York N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is generally employed in proceduresrequiring precipitation of TRICH-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encodingTRICH, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, modifications of gene expressioncan be achieved by designing complementary sequences or antisensemolecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding orregulatory regions of the gene encoding TRICH. Such technology is wellknown in the art, and antisense oligonucleotides or larger fragments canbe designed from various locations along the coding or control regionsof sequences encoding TRICH. (See, e.g., Agrawal, S., ed. (1996)Antisense Therapeutics, Humana Press Inc., Totawa N.J.)

In therapeutic use, any gene delivery system suitable for introductionof the antisense sequences into appropriate target cells can be used.Antisense sequences can be delivered intracellularly in the form of anexpression plasmid which, upon transcription, produces a sequencecomplementary to at least a portion of the cellular sequence encodingthe target protein. (See, e.g., Slater, J. E. et al. (1998) J. AllergyCli. Immunol. 102(3):469-475; and Scanlon, K. J. et al. (1995)9(13):1288-1296.) Antisense sequences can also be introducedintracellularly through the use of viral vectors, such as retrovirus andadeno-associated virus vectors. (See, e.g., Miller, A. D. (1990) Blood76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol.Ther. 63(3):323-347.) Other gene delivery mechanisms includeliposome-derived systems, artificial viral envelopes, and other systemsknown in the art. (See, e.g., Rossi, J. J. (1995) Br. Med. Bull.51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids Res.25(14):2730-2736.)

In another embodiment of the invention, polynucleotides encoding TRICHmay be used for somatic or germline gene therapy. Gene therapy may beperformed to (i) correct a genetic deficiency (e.g., in the cases ofsevere combined immunodeficiency (SCID)-X1 disease characterized byX-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science288:669-672), severe combined immunodeficiency syndrome associated withan inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al.(1995) Science 270:475-480; Bordignon, C. et al. (1995) Science270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216;Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G.et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familialhypercholesterolemia, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (HIV)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus(HBV, HCV); fungal parasites, such as Candida albicans andParacoccidioides brasiliensis; and protozoan parasites such asPlasmodium falciparum and Trypanosoma cruzi). In the case where agenetic deficiency in TRICH expression or regulation causes disease, theexpression of TRICH from an appropriate population of transduced cellsmay alleviate the clinical manifestations caused by the geneticdeficiency.

In a further embodiment of the invention, diseases or disorders causedby deficiencies in TRICH are treated by constructing mammalianexpression vectors encoding TRICH and introducing these vectors bymechanical means into TRICH-deficient cells. Mechanical transfertechnologies for use with cells in vivo or ex vitro include (i) directDNA microinjection into individual cells, (ii) ballistic gold particledelivery, (iii) liposome-mediated transfection, (iv) receptor-mediatedgene transfer, and (v) the use of DNA transposons (Morgan, R. A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell91:501-510; Boulay, J-L. and H. Récipon (1998) Curr. Opin. Biotechnol.9:445-450).

Expression vectors that may be effective for the expression of TRICHinclude, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP,PVAX vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT, PCMV-TAG,PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF, PTET-ON, PTRE2,PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). TRICH may be expressedusing (i) a constitutively active promoter, (e.g., from cytomegalovirus(CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), orβ-actin genes), (ii) an inducible promoter (e.g., thetetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin.Biotechnol. 9:451-456), commercially available in the T-REX plasmid(Invitrogen)); the ecdysone-inducible promoter (available in theplasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin induciblepromoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V.and Blau, H. M. supra)), or (iii) a tissue-specific promoter or thenative promoter of the endogenous gene encoding TRICH from a normalindividual.

Commercially available liposome transformation kits (e.g., the PERFECTLIPID TRANSFECTION KIT, available from Invitrogen) allow one withordinary skill in the art to deliver polynucleotides to target cells inculture and require minimal effort to optimize experimental parameters.In the alternative, transformation is performed using the calciumphosphate method (Graham, F. L. and A. J. Eb (1973) Virology52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J.1:841-845). The introduction of DNA to primary cells requiresmodification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused bygenetic defects with respect to TRICH expression are treated byconstructing a retrovirus vector consisting of (i) the polynucleotideencoding TRICH under the control of an independent promoter or theretrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNApackaging signals, and (iii) a Rev-responsive element (RRE) along withadditional retrovirus cis-acting RNA sequences and coding sequencesrequired for efficient vector propagation. Retrovirus vectors (e.g., PFBand PFBNEO) are commercially available (Stratagene) and are based onpublished data (Riviere, I. et al. (1995) Proc. Nati. Acad. Sci. USA92:6733-6737), incorporated by reference herein. The vector ispropagated in an appropriate vector producing cell line (VPCL) thatexpresses an envelope gene with a tropism for receptors on the targetcells or a promiscuous envelope protein such as VSVg (Armentano, D. etal. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol.61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol.62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey,R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 toRigg (“Method for obtaining retrovirus packaging cell lines producinghigh transducing efficiency retroviral supernatant”) discloses a methodfor obtaining retrovirus packaging cell lines and is hereby incorporatedby reference. Propagation of retrovirus vectors, transduction of apopulation of cells (e.g., CD4⁺ T-cells), and the return of transducedcells to a patient are procedures well known to persons skilled in theart of gene therapy and have been well documented (Ranga, U. et al.(1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U.et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)Blood 89:2283-2290).

In the alternative, an adenovirus-based gene therapy delivery system isused to deliver polynucleotides encoding TRICH to cells which have oneor more genetic abnormalities with respect to the expression of TRICH.The construction and packaging of adenovirus-based vectors are wellknown to those with ordinary skill in the art. Replication defectiveadenovirus vectors have proven to be versatile for importing genesencoding immunoregulatory proteins into intact islets in the pancreas(Csete, M. E. et al. (1995) Transplantation 27:263-268). Potentiallyuseful adenoviral vectors are described in U.S. Pat. No. 5,707,618 toArmentano (“Adenovirus vectors for gene therapy”), hereby incorporatedby reference. For adenoviral vectors, see also Antinozzi, P. A. et al.(1999) Annu. Rev. Nutr. 19:511-544 and Verma, I. M. and N. Somia (1997)Nature 18:389:239-242, both incorporated by reference herein.

In another alternative, a herpes-based, gene therapy delivery system isused to deliver polynucleotides encoding TRICH to target cells whichhave one or more genetic abnormalities with respect to the expression ofTRICH. The use of herpes simplex virus (HSV)-based vectors may beespecially valuable for introducing TRICH to cells of the centralnervous system, for which HSV has a tropism. The construction andpackaging of herpes-based vectors are well known to those with ordinaryskill in the art. A replication-competent herpes simplex virus (HSV)type 1-based vector has been used to deliver a reporter gene to the eyesof primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). Theconstruction of a HSV-1 virus vector has also been disclosed in detailin U.S. Pat. No. 5,804,413 to DeLuca (“Herpes simplex virus strains forgene transfer”), which is hereby incorporated by reference. U.S. Pat.No. 5,804,413 teaches the use of recombinant HSV d92 which consists of agenome containing at least one exogenous gene to be transferred to acell under the control of the appropriate promoter for purposesincluding human gene therapy. Also taught by this patent are theconstruction and use of recombinant HSV strains deleted for ICP4, ICP27and ICP22. For HSV vectors, see also Goins, W. F. et al. (1999) J.Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161,hereby incorporated by reference. The manipulation of cloned herpesvirussequences, the generation of recombinant virus following thetransfection of multiple plasmids containing different segments of thelarge herpesvirus genomes, the growth and propagation of herpesvirus,and the infection of cells with herpesvirus are techniques well known tothose of ordinary skill in the art.

In another alternative, an alphavirus (positive, single-stranded RNAvirus) vector is used to deliver polynucleotides encoding TRICH totarget cells. The biology of the prototypic alphavirus, Semliki ForestVirus (SFV), has been studied extensively and gene transfer vectors havebeen based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin.Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomicRNA is generated that normally encodes the viral capsid proteins. Thissubgenomic RNA replicates to higher levels than the full length genomicRNA, resulting in the overproduction of capsid proteins relative to theviral proteins with enzymatic activity (e.g., protease and polymerase).Similarly, inserting the coding sequence for TRICH into the alphavirusgenome in place of the capsid-coding region results in the production ofa large number of TRICH-coding RNAs and the synthesis of high levels ofTRICH in vector transduced cells. While alphavirus infection istypically associated with cell lysis within a few days, the ability toestablish a persistent infection in hamster normal kidney cells (BHK-21)with a variant of Sindbis virus (SIN) indicates that the lyticreplication of alphaviruses can be altered to suit the needs of the genetherapy application (Dryga, S. A. et al. (1997) Virology 228:74-83). Thewide host range of alphaviruses will allow the introduction of TRICHinto a variety of cell types. The specific transduction of a subset ofcells in a population may require the sorting of cells prior totransduction. The methods of manipulating infectious cDNA clones ofalphaviruses, performing alphavirus cDNA and RNA transfections, andperforming alphavirus infections, are well known to those with ordinaryskill in the art.

Oligonucleotides derived from the transcription initiation site, e.g.,between about positions −10 and +10 from the start site, may also beemployed to inhibit gene expression. Similarly, inhibition can beachieved using triple helix base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature. (See,e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecularand Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y.,pp.163-177.) A complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingTRICH.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding TRICH. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′and/or 3′ends of the molecule,or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

An additional embodiment of the invention encompasses a method forscreening for a compound which is effective in altering expression of apolynucleotide encoding TRICH. Compounds which may be effective inaltering expression of a specific polynucleotide may include, but arenot limited to, oligonucleotides, antisense oligonucleotides, triplehelix-forming oligonucleotides, transcription factors and otherpolypeptide transcriptional regulators, and non-macromolecular chemicalentities which are capable of interacting with specific polynucleotidesequences. Effective compounds may alter polynucleotide expression byacting as either inhibitors or promoters of polynucleotide expression.Thus, in the treatment of disorders associated with increased TRICHexpression or activity, a compound which specifically inhibitsexpression of the polynucleotide encoding TRICH may be therapeuticallyuseful, and in the treament of disorders associated with decreased TRICHexpression or activity, a compound which specifically promotesexpression of the polynucleotide encoding TRICH may be therapeuticallyuseful.

At least one, and up to a plurality, of test compounds may be screenedfor effectiveness in altering expression of a specific polynucleotide. Atest compound may be obtained by any method commonly known in the art,including chemical modification of a compound known to be effective inaltering polynucleotide expression; selection from an existing,commercially-available or proprietary library of naturally-occurring ornon-natural chemical compounds; rational design of a compound based onchemical and/or structural properties of the target polynucleotide; andselection from a library of chemical compounds created combinatoriallyor randomly. A sample comprising a polynucleotide encoding TRICH isexposed to at least one test compound thus obtained. The sample maycomprise, for example, an intact or permeabilized cell, or an in vitrocell-free or reconstituted biochemical system. Alterations in theexpression of a polynucleotide encoding TRICH are assayed by any methodcommonly known in the art. Typically, the expression of a specificnucleotide is detected by hybridization with a probe having a nucleotidesequence complementary to the sequence of the polynucleotide encodingTRICH. The amount of hybridization may be quantified, thus forming thebasis for a comparison of the expression of the polynucleotide both withand without exposure to one or more test compounds. Detection of achange in the expression of a polynucleotide exposed to a test compoundindicates that the test compound is effective in altering the expressionof the polynucleotide. A screen for a compound effective in alteringexpression of a specific polynucleotide can be carried out, for example,using a Schizosaccharomyces pombe gene expression system (Atkins, D. etal. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) NucleicAcids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L.et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particularembodiment of the present invention involves screening a combinatoriallibrary of oligonucleotides (such as deoxyribonucleotides,ribonucleotides, peptide nucleic acids, and modified oligonucleotides)for antisense activity against a specific polynucleotide sequence(Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. etal. (2000) U.S. Pat. No. 6,022,691).

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) Nat.Biotechnol. 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such ashumans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administrationof a composition which generally comprises an active ingredientformulated with a pharmaceutically acceptable excipient. Excipients mayinclude, for example, sugars, starches, celluloses, gums, and proteins.Various formulations are commonly known and are thoroughly discussed inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.). Such compositions may consist of TRICH,antibodies to TRICH, and mimetics, agonists, antagonists, or inhibitorsof TRICH.

The compositions utilized in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid ordry powder form. These compositions are generally aerosolizedimmediately prior to inhalation by the patient. In the case of smallmolecules (e.g. traditional low molecular weight organic drugs), aerosoldelivery of fast-acting formulations is well-known in the art. In thecase of macromolecules (e.g. larger peptides and proteins), recentdevelopments in the field of pulmonary delivery via the alveolar regionof the lung have enabled the practical delivery of drugs such as insulinto blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.5,997,848). Pulmonary delivery has the advantage of administrationwithout needle injection, and obviates the need for potentially toxicpenetration enhancers.

Compositions suitable for use in the invention include compositionswherein the active ingredients are contained in an effective amount toachieve the intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for directintracellular delivery of macromolecules comprising TRICH or fragmentsthereof. For example, liposome preparations containing acell-impermeable macromolecule may promote cell fusion and intracellulardelivery of the macromolecule. Alternatively, TRICH or a fragmentthereof may be joined to a short cationic N-terminal portion from theHIV Tat-1 protein. Fusion proteins thus generated have been found totransduce into the cells of all tissues, including the brain, in a mousemodel system (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example TRICH or fragments thereof, antibodies of TRICH,and agonists, antagonists or inhibitors of TRICH, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies are used to formulate a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that includes the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind TRICH may beused for the diagnosis of disorders characterized by expression ofTRICH, or in assays to monitor patients being treated with TRICH oragonists, antagonists, or inhibitors of TRICH. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for TRICH include methodswhich utilize the antibody and a label to detect TRICH in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring TRICH, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of TRICH expression. Normal or standard values for TRICHexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, for example, human subjects, withantibodies to TRICH under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, such as photometric means. Quantities of TRICH expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingTRICH may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantify gene expression in biopsied tissues in which expression ofTRICH may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of TRICH, and tomonitor regulation of TRICH levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding TRICH or closely related molecules may be used to identifynucleic acid sequences which encode TRICH. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification willdetermine whether the probe identifies only naturally occurringsequences encoding TRICH, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, and mayhave at least 50% sequence identity to any of the TRICH encodingsequences. The hybridization probes of the subject invention may be DNAor RNA and may be derived from the sequence of SEQ ID NO:14-26 or fromgenomic sequences including promoters, enhancers, and introns of theTRICH gene.

Means for producing specific hybridization probes for DNAs encodingTRICH include the cloning of polynucleotide sequences encoding TRICH orTRICH derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding TRICH may be used for the diagnosis ofdisorders associated with expression of TRICH. Examples of suchdisorders include, but are not limited to, a transport disorder such asakinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cysticfibrosis, Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Toothdisease, diabetes mellitus, diabetes insipidus, diabetic neuropathy,Duchenne muscular dystrophy, hyperkalemic periodic paralysis,normokalemic periodic paralysis, Parkinson's disease, malignanthyperthermia, multidrug resistance, myasthenia gravis, myotonicdystrophy, catatonia, tardive dyskinesia, dystonias, peripheralneuropathy, cerebral neoplasms, prostate cancer, cardiac disordersassociated with transport, e.g., angina, bradyarrythmia, tachyarrythmia,hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nemalinemyopathy, centronuclear myopathy, lipid myopathy, mitochondrialmyopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis,inclusion body myositis, infectious myositis, polymyositis, neurologicaldisorders associated with transport, e.g., Alzheimer's disease, amnesia,bipolar disorder, dementia, depression, epilepsy, Tourette's disorder,paranoid psychoses, and schizophrenia, and other disorders associatedwith transport, e.g., neurofibromatosis, postherpetic neuralgia,trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson'sdisease, cataracts, infertility, pulmonary artery stenosis,sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave'sdisease, goiter, Cushing's disease, Addison's disease, glucose-galactosemalabsorption syndrome, hypercholesterolemia, adrenoleukodystrophy,Zellweger syndrome, Menkes disease, occipital horn syndrome, von Gierkedisease, cystinuria, iminoglycinuria, Hartup disease, and Fanconidisease; a neurological disorder such as epilepsy, ischemiccerebrovascular disease, stroke, cerebral neoplasms, Alzheimer'sdisease, Pick's disease, Huntington's disease, dementia, Parkinson'sdisease and other extrapyramidal disorders, amyotrophic lateralsclerosis and other motor neuron disorders, progressive neural muscularatrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosisand other demyelinating diseases, bacterial and viral meningitis, brainabscess, subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease, prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; a muscle disorder such ascardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker'smuscular dystrophy, myotonic dystrophy, central core disease, nemalinemyopathy, centronuclear myopathy, lipid myopathy, mitochondrialmyopathy, infectious myositis, polymyositis, dermatomyositis, inclusionbody myositis, thyrotoxic myopathy, ethanol myopathy, angina,anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing'ssyndrome, hypertension, hypoglycemia, myocardial infarction, migraine,pheochromocytoma, and myopathies including encephalopathy, epilepsy,Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder,ophthalmoplegia, and acid maltase deficiency (AMD, also known as Pompe'sdisease); and an immunological disorder such as acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermaldystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma. The polynucleotide sequences encodingTRICH may be used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; in dipstick, pin, andmultiformat ELISA-like assays; and in microarrays utilizing fluids ortissues from patients to detect altered TRICH expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding TRICH may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingTRICH may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantified and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding TRICH in the sample indicatesthe presence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of TRICH, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding TRICH, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding TRICH may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding TRICH, or a fragment of a polynucleotide complementary to thepolynucleotide encoding TRICH, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide primers derived from thepolynucleotide sequences encoding TRICH may be used to detect singlenucleotide polymorphisms (SNPs). SNPs are substitutions, insertions anddeletions that are a frequent cause of inherited or acquired geneticdisease in humans. Methods of SNP detection include, but are not limitedto, single-stranded conformation polymorphism (SSCP) and fluorescentSSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from thepolynucleotide sequences encoding TRICH are used to amplify DNA usingthe polymerase chain reaction (PCR). The DNA may be derived, forexample, from diseased or normal tissue, biopsy samples, bodily fluids,and the like. SNPs in the DNA cause differences in the secondary andtertiary structures of PCR products in single-stranded form, and thesedifferences are detectable using gel electrophoresis in non-denaturinggels. In fSCCP, the oligonucleotide primers are fluorescently labeled,which allows detection of the amplimers in high-throughput equipmentsuch as DNA sequencing machines. Additionally, sequence databaseanalysis methods, termed in silico SNP (isSNP), are capable ofidentifying polymorphisms by comparing the sequence of individualoverlapping DNA fragments which assemble into a common consensussequence. These computer-based methods filter out sequence variationsdue to laboratory preparation of DNA and sequencing errors usingstatistical models and automated analyses of DNA sequence chromatograms.In the alternative, SNPs may be detected and characterized by massspectrometry using, for example, the high throughput MASSARRAY system(Sequenom, Inc., San Diego Calif.).

Methods which may also be used to quantify the expression of TRICHinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin a high-throughput format where the oligomer or polynucleotide ofinterest is presented in various dilutions and a spectrophotometric orcolorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used aselements on a microarray. The microarray can be used in transcriptimaging techniques which monitor the relative expression levels of largenumbers of genes simultaneously as described below. The microarray mayalso be used to identify genetic variants, mutations, and polymorphisms.This information may be used to determine gene function, to understandthe genetic basis of a disorder, to diagnose a disorder, to monitorprogression/regression of disease as a function of gene expression, andto develop and monitor the activities of therapeutic agents in thetreatment of disease. In particular, this information may be used todevelop a pharmacogenomic profile of a patient in order to select themost appropriate and effective treatment regimen for that patient. Forexample, therapeutic agents which are highly effective and display thefewest side effects may be selected for a patient based on his/herpharmacogenomic profile.

In another embodiment, TRICH, fragments of TRICH, or antibodies specificfor TRICH may be used as elements on a microarray. The microarray may beused to monitor or measure protein-protein interactions, drug-targetinteractions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of thepresent invention to generate a transcript image of a tissue or celltype. A transcript image represents the global pattern of geneexpression by a particular tissue or cell type. Global gene expressionpatterns are analyzed by quantifying the number of expressed genes andtheir relative abundance under given conditions and at a given time.(See Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat.No. 5,840,484, expressly incorporated by reference herein.) Thus atranscript image may be generated by hybridizing the polynucleotides ofthe present invention or their complements to the totality oftranscripts or reverse transcripts of a particular tissue or cell type.In one embodiment, the hybridization takes place in high-throughputformat, wherein the polynucleotides of the present invention or theircomplements comprise a subset of a plurality of elements on amicroarray. The resultant transcript image would provide a profile ofgene activity.

Transcript images may be generated using transcripts isolated fromtissues, cell lines, biopsies, or other biological samples. Thetranscript image may thus reflect gene expression in vivo, as in thecase of a tissue or biopsy sample, or in vitro, as in the case of a cellline.

Transcript images which profile the expression of the polynucleotides ofthe present invention may also be used in conjunction with in vitromodel systems and preclinical evaluation of pharmaceuticals, as well astoxicological testing of industrial and naturally-occurringenvironmental compounds. All compounds induce characteristic geneexpression patterns, frequently termed molecular fingerprints ortoxicant signatures, which are indicative of mechanisms of action andtoxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159;Steiner, S. and N. L. Anderson (2000) Toxicol. Lett. 112-113:467-471,expressly incorporated by reference herein). If a test compound has asignature similar to that of a compound with known toxicity, it islikely to share those toxic properties. These fingerprints or signaturesare most useful and refined when they contain expression informationfrom a large number of genes and gene families. Ideally, a genome-widemeasurement of expression provides the highest quality signature. Evengenes whose expression is not altered by any tested compounds areimportant as well, as the levels of expression of these genes are usedto normalize the rest of the expression data. The normalizationprocedure is useful for comparison of expression data after treatmentwith different compounds. While the assignment of gene function toelements of a toxicant signature aids in interpretation of toxicitymechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction oftoxicity. (See, for example, Press Release 00-02 from the NationalInstitute of Environmental Health Sciences, released Feb. 29, 2000,available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore,it is important and desirable in toxicological screening using toxicantsignatures to include all expressed gene sequences.

In one embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing nucleic acids with the testcompound. Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

Another particular embodiment relates to the use of the polypeptidesequences of the present invention to analyze the proteome of a tissueor cell type. The term proteome refers to the global pattern of proteinexpression in a particular tissue or cell type. Each protein componentof a proteome can be subjected individually to further analysis.Proteome expression patterns, or profiles, are analyzed by quantifyingthe number of expressed proteins and their relative abundance undergiven conditions and at a given time. A profile of a cell's proteome maythus be generated by separating and analyzing the polypeptides of aparticular tissue or cell type. In one embodiment, the separation isachieved using two-dimensional gel electrophoresis, in which proteinsfrom a sample are separated by isoelectric focusing in the firstdimension, and then according to molecular weight by sodium dodecylsulfate slab gel electrophoresis in the second dimension (Steiner andAnderson, supra). The proteins are visualized in the gel as discrete anduniquely positioned spots, typically by staining the gel with an agentsuch as Coomassie Blue or silver or fluorescent stains. The opticaldensity of each protein spot is generally proportional to the level ofthe protein in the sample. The optical densities of equivalentlypositioned protein spots from different samples, for example, frombiological samples either treated or untreated with a test compound ortherapeutic agent, are compared to identify any changes in protein spotdensity related to the treatment. The proteins in the spots arepartially sequenced using, for example, standard methods employingchemical or enzymatic cleavage followed by mass spectrometry. Theidentity of the protein in a spot may be determined by comparing itspartial sequence, preferably of at least 5 contiguous amino acidresidues, to the polypeptide sequences of the present invention. In somecases, further sequence data may be obtained for definitive proteinidentification.

A proteomic profile may also be generated using antibodies specific forTRICH to quantify the levels of TRICH expression. In one embodiment, theantibodies are used as elements on a microarray, and protein expressionlevels are quantified by exposing the microarray to the sample anddetecting the levels of protein bound to each array element (Lueking, A.et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999)Biotechniques 27:778-788). Detection may be performed by a variety ofmethods known in the art, for example, by reacting the proteins in thesample with a thiol- or amino-reactive fluorescent compound anddetecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins that are expressed in the treated biological sample areseparated so that the amount of each protein can be quantified. Theamount of each protein is compared to the amount of the correspondingprotein in an untreated biological sample. A difference in the amount ofprotein between the two samples is indicative of a toxic response to thetest compound in the treated sample. Individual proteins are identifiedby sequencing the amino acid residues of the individual proteins andcomparing these partial sequences to the polypeptides of the presentinvention.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins from the biological sample are incubated with antibodiesspecific to the polypeptides of the present invention. The amount ofprotein recognized by the antibodies is quantified. The amount ofprotein in the treated biological sample is compared with the amount inan untreated biological sample. A difference in the amount of proteinbetween the two samples is indicative of a toxic response to the testcompound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO095/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.) Various types ofmicroarrays are well known and thoroughly described in DNA Microarrays:A Practical Approach, M. Schena, ed. (1999) Oxford University Press,London, hereby expressly incorporated by reference.

In another embodiment of the invention, nucleic acid sequences encodingTRICH may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. Either coding or noncodingsequences may be used, and in some instances, noncoding sequences may bepreferable over coding sequences. For example, conservation of a codingsequence among members of a multi-gene family may potentially causeundesired cross hybridization during chromosomal mapping. The sequencesmay be mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome cDNA libraries. (See, e.g., Harrington, J. J. et a.(1997) Nat. Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134;and Trask. B. J. (1991) Trends Genet. 7:149-154.) Once mapped, thenucleic acid sequences of the invention may be used to develop geneticlinkage maps, for example, which correlate the inheritance of a diseasestate with the inheritance of a particular chromosome region orrestriction fragment length polymorphism (RFLP). (See, for example,Lander, E. S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA83:7353-7357.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995)in Meyers, supra, pp. 965-968.) Examples of genetic map data can befound in various scientific journals or at the Online MendelianInheritance in Man (OMIM) World Wide Web site. Correlation between thelocation of the gene encoding TRICH on a physical map and a specificdisorder, or a predisposition to a specific disorder, may help definethe region of DNA associated with that disorder and thus may furtherpositional cloning efforts.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the exact chromosomal locus is notknown. This information is valuable to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once the gene or genes responsible for a disease or syndromehave been crudely localized by genetic linkage to a particular genomicregion, e.g., ataxia-telangiectasia to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the instant invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, TRICH, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenTRICH and the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with TRICH, or fragments thereof, and washed. Bound TRICH isthen detected by methods well known in the art. Purified TRICH can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding TRICH specificallycompete with a test compound for binding TRICH. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with TRICH.

In additional embodiments, the nucleotide sequences which encode TRICHmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

The disclosures of all patents, applications, and publications mentionedabove and below, in particular U.S. Ser. No. 60/184,866, U.S. Ser. No.60/187,947, U.S. Ser. No. 60/188,333, U.S. Ser. No. 60/190,230, U.S.Ser. No. 60/192,077, and U.S. Ser. No. 60/193,500, are hereby expresslyincorporated by reference.

EXAMPLES I. Construction of cDNA Libraries

Incyte cDNAs were derived from cDNA libraries described in the LIFESEQGOLD database (Incyte Genomics, Palo Alto Calif.) and shown in Table 4,column 5. Some tissues were homogenized and lysed in guanidiniumisothiocyanate, while others were homogenized and lysed in phenol or ina suitable mixture of denaturants, such as TRIZOL (Life Technologies), amonophasic solution of phenol and guanidine isothiocyanate. Theresulting lysates were centrifuged over CsCl cushions or extracted withchloroform. RNA was precipitated from the lysates with eitherisopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A)+RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA was isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIZAP vector system (Stratagene) orSUPERSCRIPT plasmid system (Life Technologies), using the recommendedprocedures or similar methods known in the art. (See, e.g., Ausubel,1997, supra, units 5.1-6.6.) Reverse transcription was initiated usingoligo d(T) or random primers. Synthetic oligonucleotide adapters wereligated to double stranded cDNA, and the cDNA was digested with theappropriate restriction enzyme or enzymes. For most libraries, the cDNAwas size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B,or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (LifeTechnologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad Calif.), PBK-CMVplasmid (Stratagene), or pINCY (Incyte Genomics, Palo Alto Calif.), orderivatives thereof. Recombinant plasmids were transformed intocompetent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR fromStratagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered from hostcells by in vivo excision using the UNIZAP vector system (Stratagene) orby cell lysis. Plasmids were purified using at least one of thefollowing: a Magic or WIZARD Minipreps DNA purification system(Promega); an AGTC Miniprep purification kit (Edge Biosystems,Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96plasmid purification kit from QIAGEN. Following precipitation, plasmidswere resuspended in 0.1 ml of distilled water and stored, with orwithout lyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V. B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example II weresequenced as follows. Sequencing reactions were processed using standardmethods or high-throughput instrumentation such as the ABI CATALYST 800(Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJResearch) in conjunction with the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNAsequencing reactions were prepared using reagents provided by AmershamPharmacia Biotech or supplied in ABI sequencing kits such as the ABIPRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppliedBiosystems). Electrophoretic separation of cDNA sequencing reactions anddetection of labeled polynucleotides were carried out using the MEGABACE1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or377 sequencing system (Applied Biosystems) in conjunction with standardABI protocols and base calling software; or other sequence analysissystems known in the art. Reading frames within the cDNA sequences wereidentified using standard methods (reviewed in Ausubel, 1997, supra,unit 7.7). Some of the cDNA sequences were selected for extension usingthe techniques disclosed in Example VIII.

The polynucleotide sequences derived from Incyte cDNAs were validated byremoving vector, linker, and poly(A) sequences and by masking ambiguousbases, using algorithms and programs based on BLAST, dynamicprogramming, and dinucleotide nearest neighbor analysis. The Incyte cDNAsequences or translations thereof were then queried against a selectionof public databases such as the GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM,and hidden Markov model (HMM)-based protein family databases such asPFAM. (HMM is a probabilistic approach which analyzes consensus primarystructures of gene families. See, for example, Eddy, S. R. (1996) Curr.Opin. Struct. Biol. 6:361-365.) The queries were performed usingprograms based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNAsequences were assembled to produce full length polynucleotidesequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitchedsequences, stretched sequences, or Genscan-predicted coding sequences(see Examples IV and V) were used to extend Incyte cDNA assemblages tofull length. Assembly was performed using programs based on Phred,Phrap, and Consed, and cDNA assemblages were screened for open readingframes using programs based on GeneMark, BLAST, and FASTA. The fulllength polynucleotide sequences were translated to derive thecorresponding full length polypeptide sequences. Alternatively, apolypeptide of the invention may begin at any of the methionine residuesof the full length translated polypeptide. Full length polypeptidesequences were subsequently analyzed by querying against databases suchas the GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS,DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based proteinfamily databases such as PFAM. Full length polynucleotide sequences arealso analyzed using MACDNASIS PRO software (Hitachi SoftwareEngineering, South San Francisco Calif.) and LASERGENE software(DNASTAR). Polynucleotide and polypeptide sequence alignments aregenerated using default parameters specified by the CLUSTAL algorithm asincorporated into the MEGALIGN multisequence alignment program(DNASTAR), which also calculates the percent identity between alignedsequences.

Table 7 summarizes the tools, programs, and algorithms used for theanalysis and assembly of Incyte cDNA and full length sequences andprovides applicable descriptions, references, and threshold parameters.The first column of Table 7 shows the tools, programs, and algorithmsused, the second column provides brief descriptions thereof, the thirdcolumn presents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score or the lower the probability value, the greater theidentity between two sequences).

The programs described above for the assembly and analysis of fulllength polynucleotide and polypeptide sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO: 14-26.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies are described in Table 4,column 4.

IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative transporters and ion channels were initially identified byrunning the Genscan gene identification program against public genomicsequence databases (e.g., gbpri and gbhtg). Genscan is a general-purposegene identification program which analyzes genomic DNA sequences from avariety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol.268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol.8:346-354). The program concatenates predicted exons to form anassembled cDNA sequence extending from a methionine to a stop codon. Theoutput of Genscan is a FASTA database of polynucleotide and polypeptidesequences. The maximum range of sequence for Genscan to analyze at oncewas set to 30 kb. To determine which of these Genscan predicted cDNAsequences encode transporters and ion channels, the encoded polypeptideswere analyzed by querying against PFAM models for transporters and ionchannels. Potential transporters and ion channels were also identifiedby homology to Incyte cDNA sequences that had been annotated astransporters and ion channels. These selected Genscan-predictedsequences were then compared by BLAST analysis to the genpept and gbpripublic databases. Where necessary, the Genscan-predicted sequences werethen edited by comparison to the top BLAST hit from genpept to correcterrors in the sequence predicted by Genscan, such as extra or omittedexons. BLAST analysis was also used to find any Incyte cDNA or publiccDNA coverage of the Genscan-predicted sequences, thus providingevidence for transcription. When Incyte cDNA coverage was available,this information was used to correct or confirm the Genscan predictedsequence. Full length polynucleotide sequences were obtained byassembling Genscan-predicted coding sequences with Incyte cDNA sequencesand/or public cDNA sequences using the assembly process described inExample III. Alternatively, full length polynucleotide sequences werederived entirely from edited or unedited Genscan-predicted codingsequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data

“Stitched” Sequences

Partial cDNA sequences were extended with exons predicted by the Genscangene identification program described in Example IV. Partial cDNAsassembled as described in Example III were mapped to genomic DNA andparsed into clusters containing related cDNAs and Genscan exonpredictions from one or more genomic sequences. Each cluster wasanalyzed using an algorithm based on graph theory and dynamicprogramming to integrate cDNA and genomic information, generatingpossible splice variants that were subsequently confirmed, edited, orextended to create a full length sequence. Sequence intervals in whichthe entire length of the interval was present on more than one sequencein the cluster were identified, and intervals thus identified wereconsidered to be equivalent by transitivity. For example, if an intervalwas present on a cDNA and two genomic sequences, then all threeintervals were considered to be equivalent. This process allowsunrelated but consecutive genomic sequences to be brought together,bridged by cDNA sequence. Intervals thus identified were then “stitched”together by the stitching algorithm in the order that they appear alongtheir parent sequences to generate the longest possible sequence, aswell as sequence variants. Linkages between intervals which proceedalong one type of parent sequence (cDNA to cDNA or genomic sequence togenomic sequence) were given preference over linkages which changeparent type (cDNA to genomic sequence). The resultant stitched sequenceswere translated and compared by BLAST analysis to the genpept and gbpripublic databases. Incorrect exons predicted by Genscan were corrected bycomparison to the top BLAST hit from genpept. Sequences were furtherextended with additional cDNA sequences, or by inspection of genomicDNA, when necessary.

“Stretched” Sequences

Partial DNA sequences were extended to full length with an algorithmbased on BLAST analysis. First, partial cDNAs assembled as described inExample III were queried against public databases such as the GenBankprimate, rodent, mammalian, vertebrate, and eukaryote databases usingthe BLAST program. The nearest GenBank protein homolog was then comparedby BLAST analysis to either Incyte cDNA sequences or GenScan exonpredicted sequences described in Example IV. A chimeric protein wasgenerated by using the resultant high-scoring segment pairs (HSPs) tomap the translated sequences onto the GenBank protein homolog.Insertions or deletions may occur in the chimeric protein with respectto the original GenBank protein homolog. The GenBank protein homolog,the chimeric protein, or both were used as probes to search forhomologous genomic sequences from the public human genome databases.Partial DNA sequences were therefore “stretched” or extended by theaddition of homologous genomic sequences. The resultant stretchedsequences were examined to determine whether it contained a completegene.

VI. Chromosomal Mapping of TRICH Encoding Polynucleotides

The sequences which were used to assemble SEQ ID NO:14-26 were comparedwith sequences from the Incyte LIFESEQ database and public domaindatabases using BLAST and other implementations of the Smith-Watermanalgorithm. Sequences from these databases that matched SEQ ID NO:14-26were assembled into clusters of contiguous and overlapping sequencesusing assembly algorithms such as Phrap (Table 7). Radiation hybrid andgenetic mapping data available from public resources such as theStanford Human Genome Center (SHGC), Whitehead Institute for GenomeResearch (WIGR), and Généthon were used to determine if any of theclustered sequences had been previously mapped. Inclusion of a mappedsequence in a cluster resulted in the assignment of all sequences ofthat cluster, including its particular SEQ ID NO:, to that map location.

Map locations are represented by ranges, or intervals, or humanchromosomes. The map position of an interval, in centiMorgans, ismeasured relative to the terminus of the chromosome's p-arm. (ThecentiMorgan (cM) is a unit of measurement based on recombinationfrequencies between chromosomal markers. On average, 1 cM is roughlyequivalent to 1 megabase (Mb) of DNA in humans, although this can varywidely due to hot and cold spots of recombination.) The cM distances arebased on genetic markers mapped by Généthon which provide boundaries forradiation hybrid markers whose sequences were included in each of theclusters. Human genome maps and other resources available to the public,such as the NCBI “GeneMap'99” World Wide Web site(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine ifpreviously identified disease genes map within or in proximity to theintervals indicated above.

In this manner, SEQ ID NO:18 was mapped to chromosome 3 within theinterval from 136.10 to 142.20 centiMorgans.

VII. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel (1995) supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in cDNA databases such as GenBank orLIFESEQ (Incyte Genomics). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:BLAST Score×Percent Identity/5×minimum {length(Seq. 1), length(Seq. 2)}The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. The productscore is a normalized value between 0 and 100, and is calculated asfollows: the BLAST score is multiplied by the percent nucleotideidentity and the product is divided by (5 times the length of theshorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and −4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,then the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% identity and 50% overlap at one end, or 79%identity and 100% overlap.

Alternatively, polynucleotide sequences encoding TRICH are analyzed withrespect to the tissue sources from which they were derived. For example,some full length sequences are assembled, at least in part, withoverlapping Incyte cDNA sequences (see Example III). Each cDNA sequenceis derived from a cDNA library constructed from a human tissue. Eachhuman tissue is classified into one of the following organ/tissuecategories: cardiovascular system; connective tissue; digestive system;embryonic structures; endocrine system; exocrine glands; genitalia,female; genitalia, male; germ cells; hemic and immune system; liver;musculoskeletal system; nervous system; pancreas; respiratory system;sense organs; skin; stomatognathic system; unclassified/mixed; orurinary tract. The number of libraries in each category is counted anddivided by the total number of libraries across all categories.Similarly, each human tissue is classified into one of the followingdisease/condition categories: cancer, cell line, developmental,inflammation, neurological, trauma, cardiovascular, pooled, and other,and the number of libraries in each category is counted and divided bythe total number of libraries across all categories. The resultingpercentages reflect the tissue- and disease-specific expression of cDNAencoding TRICH. cDNA sequences and cDNA library/tissue information arefound in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

VII. Extension of TRICH Encoding Polynucleotides

Full length polynucleotide sequences were also produced by extension ofan appropriate fragment of the full length molecule usingoligonucleotide primers designed from this fragment. One primer wassynthesized to initiate 5′ extension of the known fragment, and theother primer was synthesized to initiate 3′ extension of the knownfragment. The initial primers were designed using OLIGO 4.06 software(National Biosciences), or another appropriate program, to be about 22to 30 nucleotides in length, to have a GC content of about 50% or more,and to anneal to the target sequence at temperatures of about 68° C. toabout 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 nmol of each primer, reaction buffer containing Mg²⁺, (NH₄)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (Amersham PharmaciaBiotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

The concentration of DNA in each well was determined by dispensing 100μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; MolecularProbes, Eugene OR) dissolved in 1× TE and 0.5 μl of undiluted PCRproduct into each well of an opaque fluorimeter plate (Corning Costar,Acton Mass.), allowing the DNA to bind to the reagent. The plate wasscanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measurethe fluorescence of the sample and to quantify the concentration of DNA.A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1 % agarose gel to determine which reactions weresuccessful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Pharmacia Biotech). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase(Stratagene) to fill-in restriction site overhangs, and transfected intocompetent E. coli cells. Transformed cells were selected onantibiotic-containing media, and individual colonies were picked andcultured overnight at 37° C. in 384-well plates in LB/2× carb liquidmedia.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethysulfoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Applied Biosystems).

In like manner, full length polynucleotide sequences are verified usingthe above procedure or are used to obtain 5′ regulatory sequences usingthe above procedure along with oligonucleotides designed for suchextension, and an appropriate genomic library.

IX. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:14-26 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ—³²P] adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under conditions of up to, for example, 0.1× saline sodiumcitrate and 0.5% sodium dodecyl sulfate. Hybridization patterns arevisualized using autoradiography or an alternative imaging means andcompared.

X. Microarrays

The linkage or synthesis of array elements upon a microarray can beachieved utilizing photolithography, piezoelectric printing (ink-jetprinting, See, e.g., Baldeschweiler, supra.), mechanical microspottingtechnologies, and derivatives thereof. The substrate in each of theaforementioned technologies should be uniform and solid with anon-porous surface (Schena (1999), supra). Suggested substrates includesilicon, silica, glass slides, glass chips, and silicon wafers.Alternatively, a procedure analogous to a dot or slot blot may also beused to arrange and link elements to the surface of a substrate usingthermal, UV, chemical, or mechanical bonding procedures. A typical arraymay be produced using available methods and machines well known to thoseof ordinary skill in the art and may contain any appropriate number ofelements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470;Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J.Hodgson (1998) Nat. Biotechnol. 16:27-31.)

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments oroligomers thereof may comprise the elements of the microarray. Fragmentsor oligomers suitable for hybridization can be selected using softwarewell known in the art such as LASERGENE software (DNASTAR). The arrayelements are hybridized with polynucleotides in a biological sample. Thepolynucleotides in the biological sample are conjugated to a fluorescentlabel or other molecular tag for ease of detection. After hybridization,nonhybridized nucleotides from the biological sample are removed, and afluorescence scanner is used to detect hybridization at each arrayelement. Alternatively, laser desorbtion and mass spectrometry may beused for detection of hybridization. The degree of complementarity andthe relative abundance of each polynucleotide which hybridizes to anelement on the microarray may be assessed. In one embodiment, microarraypreparation and usage is described in detail below.

Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT)cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21 mer), 1×first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μMdGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5(Amersham Pharmacia Biotech). The reverse transcription reaction isperformed in a 25 ml volume containing 200 ng poly(A)⁺ RNA withGEMBRIGHT kits (Incyte). Specific control poly(A)⁺ RNAs are synthesizedby in vitro transcription from non-coding yeast genomic DNA. Afterincubation at 37° C. for 2 hr, each reaction sample (one with Cy3 andanother with Cy5 labeling) is treated with 2.5 ml of 0.5 M sodiumhydroxide and incubated for 20 minutes at 85° C. to the stop thereaction and degrade the RNA. Samples are purified using two successiveCHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.(CLONTECH), Palo Alto Calif.) and after combining, both reaction samplesare ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodiumacetate, and 300 ml of 100% ethanol. The sample is then dried tocompletion using a SpeedVAC (Savant Instruments Inc., Holbrook N.Y.) andresuspended in 14 μl 5×SSC/0.2% SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements.Each array element is amplified from bacterial cells containing vectorswith cloned cDNA inserts. PCR amplification uses primers complementaryto the vector sequences flanking the cDNA insert. Array elements areamplified in thirty cycles of PCR from an initial quantity of 1-2 ng toa final quantity greater than 5 μg. Amplified array elements are thenpurified using SEPHACRYL-400 (Amersham Pharmacia Biotech).

Purified array elements are immobilized on polymer-coated glass slides.Glass microscope slides (Corning) are cleaned by ultrasound in 0.1 SDSand acetone, with extensive distilled water washes between and aftertreatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/μl, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 nl ofarray element sample per slide.

Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker(Stratagene). Microarrays are washed at room temperature once in 0.2%SDS and three times in distilled water. Non-specific binding sites areblocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2%SDS hybridization buffer. The sample mixture is heated to 65° C. for 5minutes and is aliquoted onto the microarray surface and covered with an1.8 cm² coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hours at 60° C. The arrays are washed for 10 minat 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10minutes each at 45° C. in a second wash buffer (0.1×SSC), and dried.

Detection

Reporter-labeled hybridization complexes are detected with a microscopeequipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., SantaClara Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light is focused on the array using a 20× microscope objective(Nikon, Inc., Melville N.Y.). The slide containing the array is placedon a computer-controlled X-Y stage on the microscope and raster-scannedpast the objective. The 1.8 cm×1.8 cm array used in the present exampleis scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the twofluorophores sequentially. Emitted light is split, based on wavelength,into two photomultiplier tube detectors (PMT R1477, Hamamatsu PhotonicsSystems, Bridgewater N.J.) corresponding to the two fluorophores.Appropriate filters positioned between the array and the photomultipliertubes are used to filter the signals. The emission maxima of thefluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array istypically scanned twice, one scan per fluorophore using the appropriatefilters at the laser source, although the apparatus is capable ofrecording the spectra from both fluorophores simultaneously.

The sensitivity of the scans is typically calibrated using the signalintensity generated by a cDNA control species added to the samplemixture at a known concentration. A specific location on the arraycontains a complementary DNA sequence, allowing the intensity of thesignal at that location to be correlated with a weight ratio ofhybridizing species of 1:100,000. When two samples from differentsources (e.g., representing test and control cells), each labeled with adifferent fluorophore, are hybridized to a single array for the purposeof identifying genes that are differentially expressed, the calibrationis done by labeling samples of the calibrating cDNA with the twofluorophores and adding identical amounts of each to the hybridizationmixture.

The output of the photomultiplier tube is digitized using a 12-bitRTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc.,Norwood Mass.) installed in an IBM-compatible PC computer. The digitizeddata are displayed as an image where the signal intensity is mappedusing a linear 20-color transformation to a pseudocolor scale rangingfrom blue (low signal) to red (high signal). The data is also analyzedquantitatively. Where two different fluorophores are excited andmeasured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that thesignal from each spot is centered in each element of the grid. Thefluorescence signal within each element is then integrated to obtain anumerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte).

XI. Complementary Polynucleotides

Sequences complementary to the TRICH-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring TRICH. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of TRICH. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the TRICH-encoding transcript.

XII. Expression of TRICH

Expression and purification of TRICH is achieved using bacterial orvirus-based expression systems. For expression of TRICH in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express TRICH uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof TRICH in eukaryotic cells is achieved by infecting insect ormammalian cell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding TRICH by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, TRICH is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamPharmacia Biotech). Following purification, the GST moiety can beproteolytically cleaved from TRICH at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch. 10 and 16). Purified TRICH obtained by these methods can beused directly in the assays shown in Examples XVI, XVII, and XVIII whereapplicable.

XIII. Functional Assays

TRICH function is assessed by expressing the sequences encoding TRICH atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, CarlsbadCalif.), both of which contain the cytomegalovirus promoter. 5-10 μg ofrecombinant vector are transiently transfected into a human cell line,for example, an endothelial or hematopoietic cell line, using eitherliposome formulations or electroporation. 1-2 μg of an additionalplasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP and to evaluate the apoptotic state ofthe cells and other cellular properties. FCM detects and quantifies theuptake of fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyuridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York N.Y.

The influence of TRICH on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding TRICHand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding TRICH and other genes of interestcan be analyzed by northern analysis or microarray techniques.

XIV. Production of TRICH Specific Antibodies

TRICH substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the TRICH amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubel,1995, supra, ch. 11.)

Typically, oligopeptides of about 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Applied Biosystems) using FMOCchemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide and anti-TRICH activityby, for example, binding the peptide or TRICH to a substrate, blockingwith 1% BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XV. Purification of Naturally Occurring TRICH Using Specific Antibodies

Naturally occurring or recombinant TRICH is substantially purified byimmunoaffinity chromatography using antibodies specific for TRICH. Animmunoaffinity column is constructed by covalently coupling anti-TRICHantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing TRICH are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of TRICH (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/TRICH binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andTRICH is collected.

XVI. Identification of Molecules Which Interact with TRICH

Molecules which interact with TRICH may include transporter substrates,agonists or antagonists, modulatory proteins such as Gβγ proteins(Reimann, supra) or proteins involved in TRICH localization orclustering such as MAGUKs (Craven, supra). TRICH, or biologically activefragments thereof, are labeled with ¹²⁵I Bolton-Hunter reagent. (See,e.g., Bolton A. E. and W. M. Hunter (1973) Biochem. J. 133:529-539.)Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled TRICH, washed, and any wells withlabeled TRICH complex are assayed. Data obtained using differentconcentrations of TRICH are used to calculate values for the number,affinity, and association of TRICH with the candidate molecules.

Alternatively, proteins that interact with TRICH are isolated using theyeast 2-hybrid system (Fields, S. and O. Song (1989) Nature340:245-246). TRICH, or fragments thereof, are expressed as fusionproteins with the DNA binding domain of Gal4 or lexA, and potentialinteracting proteins are expressed as fusion proteins with an activationdomain. Interactions between the TRICH fusion protein and the TRICHinteracting proteins (fusion proteins with an activation domain)reconstitute a transactivation function that is observed by expressionof a reporter gene. Yeast 2-hybrid systems are commercially available,and methods for use of the yeast 2-hybrid system with ion channelproteins are discussed in Niethammer, M. and M. Sheng (1998, Meth.Enzymol. 293:104-122).

TRICH may also be used in the PATHCALLING process (CuraGen Corp., NewHaven Conn.) which employs the yeast two-hybrid system in ahigh-throughput manner to determine all interactions between theproteins encoded by two large libraries of genes (Nandabalan, K. et al.(2000) U.S. Pat. No. 6,057,101).

Potential TRICH agonists or antagonists may be tested for activation orinhibition of TRICH ion channel activity using the assays described insection XVIII.

XVII. Demonstration of TRICH Activity

Ion channel activity of TRICH is demonstrated using anelectrophysiological assay for ion conductance. TRICH can be expressedby transforming a mammalian cell line such as COS7, HeLa or CHO with aeukaryotic expression vector encoding TRICH. Eukaryotic expressionvectors are commercially available, and the techniques to introduce theminto cells are well known to those skilled in the art. A second plasmidwhich expresses any one of a number of marker genes, such asβ-galactosidase, is co-transformed into the cells to allow rapididentification of those cells which have taken up and expressed theforeign DNA. The cells are incubated for 48-72 hours aftertransformation under conditions appropriate for the cell line to allowexpression and accumulation of TRICH and β-galactosidase.

Transformed cells expressing β-galactosidase are stained blue when asuitable colorimetric substrate is added to the culture media underconditions that are well known in the art. Stained cells are tested fordifferences in membrane conductance by electrophysiological techniquesthat are well known in the art. Untransformed cells, and/or cellstransformed with either vector sequences alone or β-galactosidasesequences alone, are used as controls and tested in parallel. Cellsexpressing TRICH will have higher anion or cation conductance relativeto control cells. The contribution of TRICH to conductance can beconfirmed by incubating the cells using antibodies specific for TRICH.The antibodies will bind to the extracellular side of TRICH, therebyblocking the pore in the ion channel, and the associated conductance.

Alternatively, ion channel activity of TRICH is measured as current flowacross a TRICH-containing Xenopus laevis oocyte membrane using thetwo-electrode voltage-clamp technique (Ishi et al., supra; Jegla, T. andL. Salkoff (1997) J. Neurosci. 17:32-44). TRICH is subcloned into anappropriate Xenopus oocyte expression vector, such as pBF, and 0.5-5 ngof mRNA is injected into mature stage IV oocytes. Injected oocytes areincubated at 18° C. for 1-5 days. Inside-out macropatches are excisedinto an intracellular solution containing 116 mM K-gluconate, 4 mM KCl,and 10 mM Hepes (pH 7.2). The intracellular solution is supplementedwith varying concentrations of the TRICH mediator, such as cAMP, cGMP,or Ca⁺² (in the form of CaCl₂), where appropriate. Electrode resistanceis set at 2-5 MΩ and electrodes are filled with the intracellularsolution lacking mediator. Experiments are performed at room temperaturefrom a holding potential of 0 mV. Voltage ramps (2.5 s) from −100 to 100mV are acquired at a sampling frequency of 500 Hz. Current measured isproportional to the activity of TRICH in the assay.

Transport activity of TRICH is assayed by measuring uptake of labeledsubstrates into Xenopus laevis oocytes. Oocytes at stages V and VI areinjected with TRICH mRNA (10 ng per oocyte) and incubated for 3 days at18° C. in OR2 medium (82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl_(2,) 1 mMMgCl_(2,) 1 mM Na₂HPO_(4,) 5 mM Hepes, 3.8 mM NaOH, 50 μg/ml gentamycin,pH 7.8) to allow expression of TRICH. Oocytes are then transferred tostandard uptake medium (100 mM NaCl, 2 mM KCl, 1 mM CaCl_(2,) 1 mMMgCl_(2,) 10 mM Hepes/Tris pH 7.5). Uptake of various substrates (e.g.,amino acids, sugars, drugs, ions, and neurotransmitters) is initiated byadding labeled substrate (e.g. radiolabeled with ³H, fluorescentlylabeled with rhodamine, etc.) to the oocytes. After incubating for 30minutes, uptake is terminated by washing the oocytes three times inNa⁺-free medium, measuring the incorporated label, and comparing withcontrols. TRICH activity is proportional to the level of internalizedlabeled substrate.

ATPase activity associated with TRICH can be measured by hydrolysis ofradiolabeled ATP-[γ-³²P], separation of the hydrolysis products bychromatographic methods, and quantitation of the recovered ³²P using ascintillation counter. The reaction mixture contains ATP-[γ-³²P] andvarying amounts of TRICH in a suitable buffer incubated at 37° C. for asuitable period of time. The reaction is terminated by acidprecipitation with trichloroacetic acid and then neutralized with base,and an aliquot of the reaction mixture is subjected to membrane orfilter paper-based chromatography to separate the reaction products. Theamount of ³²P liberated is counted in a scintillation counter. Theamount of radioactivity recovered is proportional to the ATPase activityof TRICH in the assay.

XVIII. Identification of TRICH Agonists and Antagonists

TRICH is expressed in a eukaryotic cell line such as CHO (ChineseHamster Ovary) or HEK (Human Embryonic Kidney) 293. Ion channel activityof the transformed cells is measured in the presence and absence ofcandidate agonists or antagonists. Ion channel activity is assayed usingpatch clamp methods well known in the art or as described in ExampleXVII. Alternatively, ion channel activity is assayed using fluorescenttechniques that measure ion flux across the cell membrane (Velicelebi,G. et al. (1999) Meth. Enzymol. 294:20-47; West, M. R. and C. R. Molloy(1996) Anal. Biochem. 241:51-58). These assays may be adapted forhigh-throughput screening using microplates. Changes in internal ionconcentration are measured using fluorescent dyes such as the Ca²⁺indicator Fluo-4 AM, sodium-sensitive dyes such as SBFI and sodiumgreen, or the Cl⁻ indicator MQAE (all available from Molecular Probes)in combination with the FLIPR fluorimetric plate reading system(Molecular Devices). In a more generic version of this assay, changes inmembrane potential caused by ionic flux across the plasma membrane aremeasured using oxonyl dyes such as DiBAC₄ (Molecular Probes). DiBAC₄equilibrates between the extracellular solution and cellular sitesaccording to the cellular membrane potential. The dye's fluorescenceintensity is 20-fold greater when bound to hydrophobic intracellularsites, allowing detection of DiBAC₄ entry into the cell (Gonzalez, J. E.and P. A. Negulescu (1998) Curr. Opin. Biotechnol. 9:624-631). Candidateagonists or antagonists may be selected from known ion channel agonistsor antagonists, peptide libraries, or combinatorial chemical libraries.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with certain embodiments,it should be understood that the invention as claimed should not beunduly limited to such specific embodiments. Indeed, variousmodifications of the described modes for carrying out the inventionwhich are obvious to those skilled in molecular biology or relatedfields are intended to be within the scope of the following claims.TABLE 1 Poly- Incyte Incyte Polypeptide Incyte nucleotide PolynucleotideProject ID SEQ ID NO: Polypeptide ID SEQ ID NO: ID 1845722 1 1845722CD114 1845722CB1 1866774 2 1866774CD1 15 1866774CB1 2481557 3 2481557CD1 162481557CB1 3125952 4 3125952CD1 17 3125952CB1 2284306 5 2284306CD1 182284306CB1 1621218 6 1621218CD1 19 1621218CB1 70950938 7 70950938CD1 2070950938CB1 7472477 8 7472477CD1 21 7472477CB1 2864787 9 2864787CD1 222864787CB1 4297813 10 4297813CD1 23 4297813CB1 7014403 11 7014403CD1 2470144030B1 71278849 12 71278849CD1 25 71278849CB1 6879618 13 6879618CD126 6879618CB1

TABLE 2 Polypeptide Incyte GenBank Probability SEQ ID NO: Polypeptide IDID NO: score GenBank Homolog 1 1845722CD1 g3878117 3.1e−60 Mitochondrialcarrier protein [Caenorhabditis elegans] The C. elegans SequencingConsortium (1998) Science 282: 2012-2018. 3 2481557CD1 g3879782 6.3e−53Similarity to Salmonella regulatory protein UHPC [Caenorhabditiselegans] The C. elegans Sequencing Consortium (1998) Science 282:2012-2018. 4 3125952CD1 g1353859 6.7e−12 Atx2p, manganese-traffickingprotein [Saccharomyces cerevisiae] Lin, S. J. and Culotta, V. C. (1996)Mol. Cell. Biol. 16: 6303-6312. 5 2284306CD1 g5565872 5.2e−26 Felineleukemia virus subgroup C receptor FLVCR [Homo sapiens] Tailor, C. etal. (1999) J. Virol. 73: 6500-6505. 6 1621218CD1 g3880445 5.0e−16Contains similarity to Pfam domain: PF02214 (K+ channel tetramerizationdomain) [Caenorhabditis elegans] 7 70950938CD1 g6453859 1.9e−42 Putativecarnitine/acylcarnitine translocase [Arabidopsis thaliana] 8 7472477CD1g11138056 2.0e−60 Putative Na+ dependent inorganic phosphatecotransporter [Oryza sativa] 9 2864787CD1 g2696709  1.6e−219Kidney-specific organic cation transporter-like protein [Mus musculus]Mori, K. et al. (1997) FEBS Lett. 417: 371-374. 10 4297813CD1 g111380561.0e−79 Putative Na+ dependent inorganic phosphate cotransporter [Oryzasativa] 11 7014403CD1 g5001458  4.1e−117 Putative ABC transporter[Arabidopsis thaliana] Lin, X. et al. (1999) Nature 402: 761-768. 1271278849CD1 g1066457 0.0 amiloride sensitive sodium channel deltasubunit [Homo sapiens] (Waldmann, R. et al. (1995) J. Biol. Chem. 270:27411-27414) 13 6879618CD1 g3004482  6.3e−112 putative integral membranetransport protein [Rattus norvegicus] (Schomig, E. et al. (1998) FEBSLetter 425: 79-86)

TABLE 3 Amino Potential SEQ Incyte Acid Potential Glyco- Analytical IDPolypeptide Resi- Phosphorylation sylation Signature Sequences, Methodsand NO: ID dues Sites Sites Domains and Motifs Databases 1 1845722CD1315 S108 S126 S223 N302 ADENINE NUCLEOTIDE TRANSPORTER DOMAIN:BLIMPS-PRINTS T269 PR00927B: Y259-K280, PR00927E: R161-L182, PR00927G:E273-R288 PROTEIN TRANSPORT TRANSMEMBRANE REPEAT BLAST-PRODOMMITOCHONDRION CARRIER MEMBRANE INNER MITOCHONDRIAL ADP/ATP: PD000117:K61-Q255 MITOCHONDRIAL ENERGY TRANSFER PROTEINS: BLAST-DOMODM00026|P39953|136-224: S126-K209, DM00026|P40556|180-263: S126-Y210,DM00026|P40464|129-210: L124-K207, DM00026|P38127|179-262: L124-K207Mitochondrial energy transfer protein MOTIFS domain: P42-R50Mitochondrial carrier protein domain: HMMER-PFAM Y23-L307 Mitochondrialenergy transfer protein BLIMPS-BLOCKS signature: BL00215A: I27-A51,BL00215B: I173-G185 Mitochondrial energy transfer proteins PROFILESCANsignature: N25-L78, V225-V289 MITOCHONDRIAL CARRIER PR: PR00926C:G281-E301, BLIMPS-PRINTS PR00926D: M133-Q151, PR00926E: Y82-F100,PR00926F: A231-Q253 2 1866774CD1 75 T39 S67 S64 Aminoacyl-transfer RNAsynthetases PROFILESCAN class-I signature: L26-V74 3 2481557CD1 297 S37S47 T59 N58 N266 signal cleavage domain: M1-A36 SPSCAN S230 S237 S249N293 GLPT FAMILY OF TRANSPORTERS: BLAST-DOMO S268 DM02439|P09836|1-401:L84-I239, DM02439|P37948|1-403: L84-N244 4 3125952CD1 307 S132 S232 S284N29 N241 Signal peptide: M1-G22 HMMER S83 S272 Transmembrane domains:HMMER L9-N29, Y107-I125, V174-L202 Protein GUFA transmembrane membraneBLAST-PRODOM intergenic region inner conserved similarity (Myxococcusxanthus) PD004603: D134-V266 5 2284306CD1 376 S73 S117 S162 N107Transmembrane domains: L20-I60, I183-P207, HMMER S166 Y131 S211 N292L248-N268, W342-L359 Y369 Transmembrane four family: PR00259C;BLIMPS-PRINTS R168-G198 6 1621218CD1 339 S23 S34 T39 S43 N113 Signalcleavage domain: M1-M32 SPSCAN S66 S104 T126 N170 Potassium channelsignature sequence: BLIMPS-PRINTS S139 PR00169A: Q46-T65 (P < 0.016)Ionic potassium transport channel (CIK4, BLAST-PRODOM CIK1, CIK2):PD000451: M1-E76, P = 1.4e−05 Potassium channel: DM00490|A39372|31-37:BLAST-DOMO I5-P95, P = 2.4e−07 7 70950938CD1 288 T25 T35 S88 N129 Signalcleavage domain: M1-P22 SPSCAN T171 T220 S249 Mitochondrial carrierprotein domain: MOTIFS S266 P22-L30, P119-L127, P221-M229 Mitochondrialcarrier protein domain: HMMER-PFAM M1-Y77, S98-E186, Y192-W287Mitochondrial energy transporter BLIMPS-BLOCKS signature: BL00215A:V206-Q230 Adenine nucleotide translocator [Homo BLIMPS-PRINTS sapiens]:PR00927A: P2-A14, PR00927B: Y239-R260, PR00927E: T34-F55, PR00927G:D158-R173 Mitochondrial carrier protein/adenine BLIMPS-PRINTS nucleotidetranslocator [Chlorella kessleri]: PR00926B: Y115-N129, PR00926C:G261-E281, PR00926D: L112-Q130, PR00926F: G9-Q31 Adenine nucleotidetranslocator BLAST-PRODOM [Chlorella kessleri] Mitochondrial energytransfer protein: BLAST-DOMO DM00026 8 7472477CD1 577 T25 S134 S268 N3N21 Sugar transporter motif: L251-R276 MOTIFS S274 S331 T388 N28 N476Sugar transporter motif: T144-L570 HMMER-PFAM T393 T433 S458 Ammoniumtransporter protein [E. coli] BLIMPS-BLOCKS S572 T573 BL01219E:G520-C529 Sodium phosphate carrier: BLAST-DOMODM01845|59302|P34644|Q03567|A56410| 9 2864787CD1 553 S35 S46 S107 N39N56 Sugar transport proteins: BL00216B: R434-G483 BLIMPS-BLOCKS S109S167 S282 N102 Transmembrane 4 family protein BLIMPS-BLOCKS T289 T408T526 signature: BL00421A: A195-V213 T539 Transmembrane domains:F204-M222; W357-M383 HMMER Sugar (and other) transporter: T106-V530HMMER-PFAM ORGANIC TRANSPORTERLIKE TRANSPORT BLAST-PRODOM PROTEIN, RENALIONIC TRANSPORTER: PD151320: N102-K145 (p = 6.4e−09) 10 4297813CD1 473T11 T16 T30 S31 N372 Sugar transport proteins signature 2: MOTIFS S164S170 S227 L147-R172 T284 T289 T329 Sugar (and other) transporter:E42-L466 HMMER-PFAM S354 S468 T469 PHOSPHATE TRANSPORT DOMAIN; SODIUM;BLAST-DOMO RENAL: DM01845|I59302|222-505: V193-R463;DM01845|P34644|215-507: G182-D472; DM01845|Q03567|156-455: A181-Q462;DM01845|A56410|183-464: V193-D465 11 7014403CD1 598 S24 T127 S194 N23N65 ATP-BINDING TRANSPORT PROTEIN FAMILY (ABC BLIMPS-PRODOM T229 T312S390 N536 TRANSPORTERS): PD00131A: G128-D137; S407 S418 T442 PD00131B:S390-V443; PD00131C: N536-R573 S552 T571 ABC TRANSPORTER PROTEINPD002040: L422-D478; BLAST-PRODOM ABC Transporter motif: L488-A501MOTIFS ATP/GTP-binding site motif A (P-loop): MOTIFS G386-S393 ABCTRANSPORTERS FAMILY BLAST-DOMO |DM00008|P39109|1272-1482: V352-G561;|DM00008|Q10185|1239-1448: V352-G561; |DM00008|P33527|1293-1502:V352-G561; |DM00008|S64757|1302-1528: V352-M474 Transmembrane domain:F147-G167 HMMER ABC transporter transmembrane region: HMMER-PFAMW10-L304, G379-G561 ABC transporters family: BL00211A: I384-L395;BLIMPS-BLOCKS BL00211B: L488-D519 ABC transporters family signature:E469-D519 PROFILESCAN Probable GTP-binding protein: PR00326A:BLIMPS-PRINTS L382-L402 12 71278849CD1 669 S86 S154 S375 N97 N197Transmembrane domain: HMMER S385 S454 T473 N242 W119-F138 T479 T497 S502N415 Amiloride-sensitive sodium channel: HMMER_PFAM S522 S614 T163F94-L581 T244 T512 S543 Amiloride-sensitive sodium channel:BLIMPS_BLOCKS S573 T629 S653 BL01206A: F93-A103, BL01206B: Y314-Q327S173 Y194 Y550 BL01206C: G330-P348, BL01206D: A354-T402 BL01206E:Y418-P444, BL01206F: C482-S502 BL01206G: I532-L577 Amiloride-sensitivesodium channel: BLIMPS_PRINTS PR01078A: T116-Q133, PR01078B: E155-R171PR01078C: V291-Q302, PR01078D: Q305-D321 PR01078E: G330-P348, PR01078F:G355-S373 PR01078G: S385-C401, PR01078H: Y418-Q429 PR01078I: Q429-P446,PR01078J: C482-S502 PR01078K: R526-E546, PR01078L: E546-G560 PR01078M:G560-E576 AMILORIDESENSITIVE SUBUNIT NA+ CHANNEL BLAST_PRODOM PD001186:Y250-D583 EPITHELIAL SODIUM DELTA SUBUNIT BLAST_PRODOMAMILORIDESENSITIVE ION TRANSPORT CHANNEL PD040282: T588-T669 PD040286:Q53-F93 SODIUM SENSITIVE AMILORIDE; CHANNEL BLAST_DOMODM01114|P51172|38-575: P69-S607 DM01114|A49585|37-598: R235-W600DM01114|P37088|37-598: R235-W600 DM01114|P55270|17-579: R235-W600 136879618CD1 566 S104 S106 S320 N39 N56 Transmembrane domain: HMMER S327S333 T539 N99 I148-Y165 T65 S164 T224 Sugar (and other) transporter:HMMER_PFAM S225 S279 T444 T103-L543 S545 Y313 Transmembranecotransporter: BLIMPS_PRODOM PD01941E: Q139-T185 (P < 0.0088;score/strength = 0.59)

TABLE 4 Incyte Polynucleotide Polynucleotide Sequence Selected 5′ 3′ SEQID NO: ID Length Fragment(s) Sequence Fragments Position Position 141845722CB1 3599   1-835, 1581463F6 (DUODNOT01) 2379 2958  3348-3599,2894401F6 (KIDNTUT14) 991 1446 1559-2126 6287978H2 (EPIPUNA01) 209 889806154R1 (BSTMNOT01) 2194 2716 6608558T1 (HNT2TXC01) 1435 2114 5608521H1(MONOTXS05) 1197 1482 1003654H1 (BRSTNOT03) 1 227 1845722R6 (COLNNOT09)631 1179 6536640H1 (OVARDIN02) 2837 3599 1845722T6 (COLNNOT09) 1998 26004857175T6 (BRSTTUT22) 1585 2145 15 1866774CB1 3177    1-1334, 70702747V11422 2065 1801-2603 7027152H1 (LIVRNOT21) 779 1387 70700094V1 2052 26756594126J1 (LUNGFER02) 1251 1918 70701664V1 2571 3177 7055202H1(BRALNON02) 273 993 6986754H1 (BRAIFER05) 1 532 7244508H1 (PROSTMY01)1953 2577 16 2481557CB1 1117  791-902, 70623747V1 1 601   1-126,6473332H1 (PLACFEB01) 454 1117 1028-1117 17 3125952CB1 5397  3751-3967,70558367V1 3568 4169    1-3175, 70557865V1 4780 5397 4499-4615 6800092H1(COLENOR03) 1697 2382 60148633B2 3018 3547 3615912H1 (EPIPNOT01) 24992807 4306278H1 (GBLADIT01) 1122 1359 2641087F6 (LUNGTUT08) 2640 32021671905F6 (BLADNOT05) 1242 1713 3944057H1 (SCORNOT04) 2594 286970558550V1 4081 4753 1644485F6 (HEARFET01) 3443 4038 423640H1(CARCTXT01) 1 280 5764760H1 (PROSBPT02) 37 665 6467129H1 (PLACFEB01) 4771158 60148929D2 2110 2543 70558366V1 4158 4842 18 2284306CB1 1728  836-1156, 6382729T8 (FIBRUNT02) 993 1714  1-275 6730828H1 (COLITUT02)372 1014 364139T6 (PROSNOT01) 1240 1728 71117895V1 647 1179 4556661F8(KERAUNT01) 1 608 19 1621218CB1 3343  835-877, 71159478V1 1707 2291 1072-1989, 6766753J1 (BRAUNOR01) 1 508  2368-2603, 71162389V1 2320 3021 281-640, 71159042V1 2223 2869 3255-3343 7069383H1 (BRAUTDR02) 2720 3343FL1621218_00001 63 2285 20 70950938CB1 1624  786-1624 71302454V1 9791624 71300807V1 423 1093 71153235V1 1 398 FL70950938_00001 84 1018 217472477CB1 1845  1609-1638, GNN.g6624821_028 1 1845   1-333,   795-1153,463-537 22 2864787CB1 2455 668-998 5394627F6 (KIDNNOT32) 1255 1798GNN.g6648132_024 65 1611 GBI.g6562971.raw 1 403 5811218T6 (KIDCTMT02)1862 2455 6844987H1 (KIDNTMN03) 1687 2382 23 4297813CB1 1638  574-932,7276729H2 (LIVRNOS02) 918 1387   1-94, 71225004V1 1410 1638 1388-14174293939F8 (SCOMDIT01) 365 845 GNN.g6016939_000016_002. 322 1624 edit6908014J1 (PITUDIR01) 1 790 24 7014403CB1 2977  1-812 1972863F6(UCMCL5T01) 2476 2976 70169954V1 1885 2363 809631R6 (LUNGNOT04) 969 15942518163F6 (BRAITUT21) 1288 1878 6370706H1 (ENDIUNT01) 863 1210 2715384T6(THYRNOT09) 2328 2958 4114919F6 (UTRSTUT07) 2740 2977 6811774H1(ADRETUR01) 1 661 60209106U1 595 1035 7016964V1 1758 2295 25 71278849CB12714  1151-1439, 1933093F6 (COLNNOT16) 1573 2073  1-758 1874189F6(LEUKNOT02) 2176 2714 71280458V1 313 831 68020718J1 (SINTNOR01) 17112260 6308468H1 (NERDTDN03) 794 1427 71280920V1 609 964 71278849V1 1 4876147651H1 (BRANDIT03) 1043 1625 26 6879618CB1 2047  1170-1606, 3671385H1(KIDNTUT16) 1 281  1-345 3629554H1 (COLNNOT38) 272 515 6879618H1(PLACNOR01) 472 1058 GNN.g6552763_020.edit 347 2047

TABLE 5 Polynucleotide Incyte Representative SEQ ID NO: Project IDLibrary 14 1845722CB1 BRSTTUT01 15 1866774CB1 SKINBIT01 16 2481557CB1BRSTTMC01 17 3125952CB1 HUVELPB01 18 2284306CB1 STOMFET01 19 1621218CB1EOSIHET02 20 70950938CB1 BMARNOR02 22 2864787CB1 KIDNNOT20 23 4297813CB1COLNPOT01 24 7014403CB1 FIBRTXS07 25 71278849CB1 FIBPFEN06 26 6879618CB1PLACNOR01

TABLE 6 Library Vector Library Description BMARNOR02 PBLUESCRIPT Librarywas constructed using RNA isolated from the bone marrow of 24 male andfemale Caucasian donors, 16 to 70 years old. (RNA came from Clontech.)BRSTTMC01 pINCY This large size-fractionated library was constructedusing pooled cDNA from four donors. cDNA was generated using mRNAisolated from diseased breast tissue removed from a 40-year-oldCaucasian female (donor A) during a bilateral reduction mammoplasty;from breast tissue removed from a 46- year-old Caucasian female (donorB) during unilateral extended simple mastectomy with breastreconstruction; from breast tissue removed from a 56-year-old Caucasianfemale (donor C) during unilateral extended simple mastectomy with openbreast biopsy; and from breast tissue removed from a 57-year-oldCaucasian female (donor D) during a unilateral extended simplemastectomy. Pathology indicated bilateral mild fibrocystic andproliferative changes (A); deep fascia was negative for tumor (B); non-proliferative fibrocystic change (C); and benign fat replaced breastparenchyma (D). Pathology for the matched tumor tissue (B) indicatedinvasive grade 3 adenocarcinoma, ductal type, with apocrine features.Pathology for the matched tumor tissue (C) indicated invasive grade 3ductal adenocarcinoma. Pathology for the matched tumor tissue (D)indicated residual microscopic infiltrating grade 3 ductaladenocarcinoma and extensive grade 2 intraductal carcinoma. Patienthistory included breast hypertrophy and pure hypercholesterolemia (A);breast cancer (B); chronic airway obstruction and emphysema (C); andbenign hypertension, hyperlipidemia, cardiac dysrhythmia, a benign colonneoplasm, a solitary breast cyst, and a breast neoplasm of uncertainbehavior (D). Previous surgeries included open breast biopsy (B). DonorB's medications included Cytoxan and Adriamycin. BRSTTUT01 PSPORT1Library was constructed using RNA isolated from breast tumor tissueremoved from a 55-year-old Caucasian female during a unilateral extendedsimple mastectomy. Pathology indicated invasive grade 4 mammaryadenocarcinoma of mixed lobular and ductal type, extensively involvingthe left breast. The tumor was identified in the deep dermis near thelactiferous ducts with extracapsular extension. Seven mid and low andfive high axillary lymph nodes were positive for tumor. Proliferativefibrocysytic changes were characterized by apocrine metaplasia,sclerosing. adenosis, cyst formation, and ductal hyperplasia withoutatypia. Patient history included atrial tachycardia, blood in the stool,and a benign breast neoplasm. Family history included benignhypertension, atherosclerotic coronary artery disease, cerebrovasculardisease, and depressive disorder. COLNPOT01 pINCY Library wasconstructed using RNA isolated from colon polyp tissue removed from a40-year-old Caucasian female during a total colectomy. Pathologyindicated an inflammatory pseudopolyp; this tissue was associated with afocally invasive grade 2 adenocarcinoma and multiple tubuvillousadenomas. Patient history included a benign neoplasm of the bowel.EOSIHET02 PBLUESCRIPT Library was constructed using RNA isolated fromperipheral blood cells apheresed from a 48-year-old Caucasian male.Patient history included hypereosinophilia. The cell population wasdetermined to be greater than 77% eosinophils by Wright's staining.FIBPFEN06 pINCY This normalized prostate stromal fibroblast tissuelibrary was constructed from 1.56 million independent clones from aprostate fibroblast library. Starting RNA was made from fibroblasts ofprostate stroma removed from a male fetus, who died after 26 weeks'gestation. The library was normalized in two rounds using conditionsadapted from Soares et al., PNAS (1994) 91: 9228 and Bonaldo et al.,Genome Research (1996) 6: 791, except that a significantly longer(48-hours/round)reannealing hybridization was used. FIBRTXS07 pINCY Thissubtracted library was constructed using 1.3 million clones from adermal fibroblast library and was subjected to two rounds of subtractionhybridization with 2.8 million clones from an untreated dermalfibroblast tissue library. The starting library for subtraction wasconstructed using RNA isolated from treated dermal fibroblast tissueremoved from the breast of a 31-year-old Caucasian female. The cellswere treated with 9CIS retinoic acid. The hybridization probe forsubtraction was derived from a similarly constructed library from RNAisolated from untreated dermal fibroblast tissue from the same donor.Subtractive hybridization conditions were based on the methodologies ofSwaroop et al., NAR (1991) 19: 1954 and Bonaldo, et al., Genome Research(1996) 6: 791. HUVELPB01 PBLUESCRIPT Library was constructed using RNAisolated from HUV-EC-C (ATCC CRL 1730) cells that were stimulated withcytokine/LPS. RNA was isolated from two pools of HUV-EC-C cells that hadbeen treated with either gamma IFN and TNF-alpha or IL-1 beta and LPS.In the first instance, HUV-EC-C cells were treated with 4 units/ml TNFand 2 units/ml IFNg for 96 hours. In the second instance, cells weretreated with 1 units/ml IL-1 and 100 ng/ml LPS for 5 hours. KIDNNOT20pINCY Library was constructed using RNA isolated from left kidney tissueremoved from a 43-year-old Caucasian male during nephroureterectomy,regional lymph node excision, and unilateral left adrenalectomy.Pathology for the associated tumor tissue indicated a grade 2 renal cellcarcinoma. Family history included atherosclerotic coronary arterydisease. PLACNOR01 PCDNA2.1 This random primed library was constructedusing pooled cDNA from two different donors. cDNA was generated usingmRNA isolated from placental tissue removed from a Caucasian fetus(donor A), who died after 16 weeks' gestation from fetal demise andhydrocephalus and from placental tissue removed from a Caucasian malefetus (donor B), who died after 18 weeks' gestation from fetal demise.Patient history for donor A included umbilical cord wrapped around thehead (3 times) and the shoulders (1 time). Serology was positive foranti-CMV and remaining serologies were negative. Family history includedmultiple pregnancies and live births, and an abortion in the mother.Serology was negative for donor B. SKINBIT01 pINCY Library wasconstructed using RNA isolated from diseased skin tissue of the leftlower leg. Patient history included erythema nodosum of the left lowerleg. STOMFET01 pINCY Library was constructed using RNA isolated from thestomach tissue of a Caucasian female fetus, who died at 20 weeks'gestation.

TABLE 7 Program Description Reference ABI FACTURA A program that removesvector sequences and Applied Biosystems, Foster City, CA. masksambiguous bases in nucleic acid sequences. ABI/ A Fast Data Finderuseful in comparing and Applied Biosystems, Foster City, CA; PARACEL FDFannotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena,CA. ABI A program that assembles nucleic acid sequences. AppliedBiosystems, Foster City, CA. AutoAssembler BLAST A Basic Local AlignmentSearch Tool useful in Altschul, S. F. et al. (1990) J. Mol. Biol.sequence similarity search for amino acid and 215: 403-410; Altschul, S.F. et al. (1997) nucleic acid sequences. BLAST includes five NucleicAcids Res. 25: 3389-3402. functions: blastp, blastn, blastx, tblastn,and tblastx. FASTA A Pearson and Lipman algorithm that searches forPearson, W. R. and D. J. Lipman (1988) Proc. similarity between a querysequence and a group of Natl. Acad Sci. USA 85: 2444-2448; Pearson, W.R. sequences of the same type. FASTA comprises as (1990) MethodsEnzymol. 183: 63-98; least five functions: fasta, tfasta, fastx, tfastx,and and Smith, T. F. and M. S. Waterman (1981) ssearch. Adv. Appl. Math.2: 482-489. BLIMPS A BLocks IMProved Searcher that matches a Henikoff,S. and J. G. Henikoff (1991) Nucleic sequence against those in BLOCKS,PRINTS, Acids Res. 19: 6565-6572; Henikoff, J. G. and DOMO, PRODOM, andPFAM databases to search S. Henikoff (1996) Methods Enzymol. for genefamilies, sequence homology, and structural 266: 88-105; and Attwood, T.K. et al. (1997) J. fingerprint regions. Chem. Inf. Comput. Sci. 37:417-424. HMMER An algorithm for searching a query sequence againstKrogh, A. et al. (1994) J. Mol. Biol. hidden Markov model (HMM)-baseddatabases of 235: 1501-1531; Sonnhammer, E. L. L. et al. protein familyconsensus sequences, such as PFAM. (1988) Nucleic Acids Res. 26:320-322; Durbin, R. et al. (1998) Our World View, in a Nutshell,Cambridge Univ. Press, pp. 1-350. ProfileScan An algorithm that searchesfor structural and sequence Gribskov, M. et al. (1988) CABIOS 4: 61-66;motifs in protein sequences that match sequence patterns Gribskov, M. etal. (1989) Methods Enzymol. defined in Prosite. 183: 146-159; Bairoch,A. et al. (1997) Nucleic Acids Res. 25: 217-221. Phred A base-callingalgorithm that examines automated Ewing, B. et al. (1998) Genome Res.sequencer traces with high sensitivity and probability. 8: 175-185;Ewing, B. and P. Green (1998) Genome Res. 8: 186-194. Phrap A PhilsRevised Assembly Program including SWAT and Smith, T. F. and M. S.Waterman (1981) Adv. CrossMatch, programs based on efficientimplementation Appl. Math. 2: 482-489; Smith, T. F. and M. S. Watermanof the Smith-Waterman algorithm, useful in searching (1981) J. Mol.Biol. 147: 195-197; sequence homology and assembling DNA sequences. andGreen, P., University of Washington, Seattle, WA. Consed A graphicaltool for viewing and editing Phrap assemblies. Gordon, D. et al. (1998)Genome Res. 8: 195-202. SPScan A weight matrix analysis program thatscans protein Nielson, H. et al. (1997) Protein Engineering sequencesfor the presence of secretory signal peptides. 10: 1-6; Claverie, J. M.and S. Audic (1997) CABIOS 12: 431-439. TMAP A program that uses weightmatrices to delineate Persson, B. and P. Argos (1994) J. Mol. Biol.transmembrane segments on protein sequences and 237: 182-192; Persson,B. and P. Argos (1996) determine orientation. Protein Sci. 5: 363-371.TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer,E. L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segmentson protein sequences Conf. on Intelligent Systems for Mol. Biol., anddetermine orientation. Glasgow et al., eds., The Am. Assoc. forArtificial Intelligence Press, Menlo Park, CA, pp. 175-182. Motifs Aprogram that searches amino acid sequences for patterns Bairoch, A. etal. (1997) Nucleic Acids Res. 25: 217-221; that matched those defined inProsite. Wisconsin Package Program Manual, version 9, page M51-59,Genetics Computer Group, Madison, WI. Program Parameter Threshold ABIFACTURA ABI/PARACEL FDF Mismatch <50% ABI AutoAssembler BLAST ESTs:Probability value = 1.0E−8 or less Full Length sequences: Probabilityvalue = 1.0E−10 or less FASTA ESTs: fasta E value = 1.06E−6 AssembledESTs: fasta Identity = 95% or greater and Match length = 200 bases orgreater; fastx E value = 1.0E−8 or less Full Length sequences: fastxscore = 100 or greater BLIMPS Probability value = 1.0E−3 or less HMMERPFAM hits: Probability value = 1.0E−3 or less Signal peptide hits: Score= 0 or greater ProfileScan Normalized quality score ≧ GCG-specified“HIGH” value for that particular Prosite motif. Generally, score =1.4-2.1. Phred Phrap Score = 120 or greater; Match length = 56 orgreater Consed SPScan Score = 3.5 or greater TMAP TMHMMER Motifs

1. An isolated polypeptide comprising an amino acid sequence selectedfrom the group consisting of: a) an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-13, b) a naturally occurring aminoacid sequence having at least 90% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:1-13, c) abiologically active fragment of an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of anamino acid sequence selected from the group consisting of SEQ IDNO:1-13.
 2. An isolated polypeptide of claim 1 selected from the groupconsisting of SEQ ID NO:1-13.
 3. An isolated polynucleotide encoding apolypeptide of claim
 1. 4. An isolated polynucleotide encoding apolypeptide of claim
 2. 5. An isolated polynucleotide of claim 4selected from the group consisting of SEQ ID NO:14-26.
 6. A recombinantpolynucleotide comprising a promoter sequence operably linked to apolynucleotide of claim
 3. 7. A cell transformed with a recombinantpolynucleotide of claim
 6. 8. A transgenic organism comprising arecombinant polynucleotide of claim
 6. 9. A method for producing apolypeptide of claim 1, the method comprising: a) culturing a cell underconditions suitable for expression of the polypeptide, wherein said cellis transformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. An isolated antibody whichspecifically binds to a polypeptide of claim
 1. 11. An isolatedpolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of: a) a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:14-26, b) a naturally occurringpolynucleotide sequence having at least 90% sequence identity to apolynucleotide sequence selected from the group consisting of SEQ ID NO:14-26, c) a polynucleotide sequence complementary to a), d) apolynucleotide sequence complementary to b), and e) an RNA equivalent ofa)-d).
 12. An isolated polynucleotide comprising at least 60 contiguousnucleotides of a polynucleotide of claim
 11. 13. A method for detectinga target polynucleotide in a sample, said target polynucleotide having asequence of a polynucleotide of claim 11, the method comprising: a)hybridizing the sample with a probe comprising at least 20 contiguousnucleotides comprising a sequence complementary to said targetpolynucleotide in the sample, and which probe specifically hybridizes tosaid target polynucleotide, under conditions whereby a hybridizationcomplex is formed between said probe and said target polynucleotide orfragments thereof, and b) detecting the presence or absence of saidhybridization complex, and, optionally, if present, the amount thereof.14. A method of claim 13, wherein the probe comprises at least 60contiguous nucleotides.
 15. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 11, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent the amount thereof.
 16. A composition comprising an effectiveamount of a polypeptide of claim 1 and a pharmaceutically acceptableexcipient.
 17. A composition of claim 16, wherein the polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:1-13.
 18. A method for treating a disease or conditionassociated with decreased expression of functional TRICH, comprisingadministering to a patient in need of such treatment the composition ofclaim
 16. 19. A method for screening a compound for effectiveness as anagonist of a polypeptide of claim 1, the method comprising: a) exposinga sample comprising a polypeptide of claim 1 to a compound, and b)detecting agonist activity in the sample.
 20. A composition comprisingan agonist compound identified by a method of claim 19 and apharmaceutically acceptable excipient.
 21. A method for treating adisease or condition associated with decreased expression of functionalTRICH, comprising administering to a patient in need of such treatment acomposition of claim
 20. 22. A method for screening a compound foreffectiveness as an antagonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting antagonist activity in the sample.
 23. Acomposition comprising an antagonist compound identified by a method ofclaim 22 and a pharmaceutically acceptable excipient.
 24. A method fortreating a disease or condition associated with overexpression offunctional TRICH, comprising administering to a patient in need of suchtreatment a composition of claim
 23. 25. A method of screening for acompound that specifically binds to the polypeptide of claim 1, saidmethod comprising the steps of: a) combining the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 26. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, said methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 27. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 28. A method for assessing toxicity of atest compound, said method comprising: a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 11 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 11 or fragment thereof; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound. amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 29. Amethod of claim 9, wherein the polypeptide has the sequence of SEQ IDNO:1.
 30. A method of claim 9, wherein the polypeptide has the sequenceof SEQ ID NO:2.
 31. A method of claim 9, wherein the polypeptide has thesequence of SEQ ID NO:3.
 32. A method of claim 9, wherein thepolypeptide has the sequence of SEQ ID NO:4.
 33. A method of claim 9,wherein the polypeptide has the sequence of SEQ ID NO:5.
 34. A method ofclaim 9, wherein the polypeptide has the sequence of SEQ ID NO:6.
 35. Amethod of claim 9, wherein the polypeptide has the sequence of SEQ IDNO:7.
 36. A method of claim 9, wherein the polypeptide has the sequenceof SEQ ID NO:8.
 37. A method of claim 9, wherein the polypeptide has thesequence of SEQ ID NO:9.
 38. A method of claim 9, wherein thepolypeptide has the sequence of SEQ ID NO:10.
 39. A method of claim 9,wherein the polypeptide has the sequence of SEQ ID NO:11.
 40. A methodof claim 9, wherein the polypeptide has the sequence of SEQ ID NO:12.41. A method of claim 9, wherein the polypeptide has the sequence of SEQID NO:13.
 42. A diagnostic test for a condition or disease associatedwith the expression of human transporters and ion channels (TRICH) in abiological sample comprising the steps of: a) combining the biologicalsample with an antibody of claim 10, under conditions suitable for theantibody to bind the polypeptide and form an antibody:polypeptidecomplex; and b) detecting the complex, wherein the presence of thecomplex correlates with the presence of the polypeptide in thebiological sample.
 43. The antibody of claim 10, wherein the antibodyis: a) a chimeric antibody, b) a single chain antibody, c) a Fabfragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 44. Acomposition comprising an antibody of claim 10 and an acceptableexcipient.
 45. A method of diagnosing a condition or disease associatedwith the expression of human transporters and ion channels (TRICH) in asubject, comprising administering to said subject an effective amount ofthe composition of claim
 44. 46. A composition of claim 44, wherein theantibody is labeled.
 47. A method of diagnosing a condition or diseaseassociated with the expression of human transporters and ion channels(TRICH) in a subject, comprising administering to said subject aneffective amount of the composition of claim
 46. 48. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 10 comprising: a) immunizing an animal with a polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNO:1-13, or an immunogenic fragment thereof, under conditions to elicitan antibody response; b) isolating antibodies from said animal; and c)screening the isolated antibodies with the polypeptide, therebyidentifying a polyclonal antibody which binds specifically to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13.
 49. An antibody produced by a method ofclaim
 48. 50. A composition comprising the antibody of claim 49 and asuitable carrier.
 51. A method of making a monoclonal antibody with thespecificity of the antibody of claim 10 comprising: a) immunizing ananimal with a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-13, or an immunogenic fragmentthereof, under conditions to elicit an antibody response; b) isolatingantibody producing cells from the animal; c) fusing the antibodyproducing cells with immortalized cells to form monoclonalantibody-producing hybridoma cells; d) culturing the hybridoma cells;and e) isolating from the culture monoclonal antibody which bindsspecifically to a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-
 13. 52. A monoclonal antibodyproduced by a method of claim
 51. 53. A composition comprising theantibody of claim 52 and a suitable carrier.
 54. The antibody of claim10, wherein the antibody is produced by screening ng a Fab expressionlibrary.
 55. The antibody of claim 10, wherein the antibody is producedby screening a recombinant immunoglobulin library.
 56. A method fordetecting a polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-13 in a sample, comprising the steps of:a) incubating the antibody of claim 10 with a sample under conditions toallow specific binding of the antibody and the polypeptide; and b)detecting specific binding, wherein specific binding indicates thepresence of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-13 in the sample.
 57. A method ofpurifying a polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-13 from a sample, the method comprising:a) incubating the antibody of claim 10 with a sample under conditions toallow specific binding of the antibody and the polypeptide; and b)separating the antibody from the sample and obtaining the purifiedpolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-13.
 58. A microarray wherein at least oneelement of the microarray is a polynucleotide of claim
 12. 59. A methodfor generating a transcript image of a sample which containspolynucleotides, the method comprising the steps of: a) labeling thepolynucleotides of the sample, b) contacting the elements of themicroarray of claim 58 with the labeled polynucleotides of the sampleunder conditions suitable for the formation of a hybridization complex,and c) quantifying the expression of the polynucleotides in the sample.60. An array comprising different nucleotide molecules affixed indistinct physical locations on a solid substrate, wherein at least oneof said nucleotide molecules comprises a first oligonucleotide orpolynucleotide sequence specifically hybridizable with at least 30contiguous nucleotides of a target polynucleotide, said targetpolynucleotide having a sequence of claim
 11. 61. An array of claim 60,wherein said first oligonucleotide or polynucleotide sequence iscompletely complementary to at least 30 contiguous nucleotides of saidtarget polynucleotide.
 62. An array of claim 60, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 60 contiguous nucleotides of said target polynucleotide. 63.An array of claim 60, which is a microarray.
 64. An array of claim 60,further comprising said target polynucleotide hybridized to said firstoligonucleotide or polynucleotide.
 65. An array of claim 60, wherein alinker joins at least one of said nucleotide molecules to said solidsubstrate.
 66. An array of claim 60, wherein each distinct physicallocation on the substrate contains multiple nucleotide molecules havingthe same sequence, and each distinct physical location on the substratecontains nucleotide molecules having a sequence which differs from thesequence of nucleotide molecules at another physical location on thesubstrate.
 67. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 68. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:2.
 69. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:3.
 70. A polypeptide ofclaim 1, comprising the amino acid sequence of SEQ ID NO:4.
 71. Apolypeptide of claim 1, comprising the amino acid sequence of SEQ IDNO:5.
 72. A polypeptide of claim 1, comprising the amino acid sequenceof SEQ ID NO:6.
 73. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:7.
 74. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:8.
 75. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:9.
 76. A polypeptide ofclaim 1, comprising the amino acid sequence of SEQ ID NO:10.
 77. Apolypeptide of claim 1, comprising the amino acid sequence of SEQ IDNO:11.
 78. A polypeptide of claim 1, comprising the amino acid sequenceof SEQ ID NO:12.
 79. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:13.
 80. A polynucleotide of claim 11, comprisingthe polynucleotide sequence of SEQ ID NO:14.
 81. A polynucleotide ofclaim 11, comprising the polynucleotide sequence of SEQ ID NO:15.
 82. Apolynucleotide of claim 11, comprising the polynucleotide sequence ofSEQ ID NO:16.
 83. A polynucleotide of claim 11, comprising thepolynucleotide sequence of SEQ ID NO:17.
 84. A polynucleotide of claim11, comprising the polynucleotide sequence of SEQ ID NO:18.
 85. Apolynucleotide of claim 11, comprising the polynucleotide sequence ofSEQ ID NO:19.
 86. A polynucleotide of claim 11, comprising thepolynucleotide sequence of SEQ ID NO:20.
 87. A polynucleotide of claim11, comprising the polynucleotide sequence of SEQ ID NO:21.
 88. Apolynucleotide of claim 11, comprising the polynucleotide sequence ofSEQ ID NO:22.
 89. A polynucleotide of claim 11, comprising thepolynucleotide sequence of SEQ ID NO:23.
 90. A polynucleotide of claim11, comprising the polynucleotide sequence of SEQ ID NO:24.
 91. Apolynucleotide of claim 11, comprising the polynucleotide sequence ofSEQ ID NO:25.
 92. A polynucleotide of claim 11, comprising thepolynucleotide sequence of SEQ ID NO:26.